Reagents Useful for Synthesizing Rhodamine-Labeled Oligonucleotides

ABSTRACT

The present disclosure provides reagents that can be used to label synthetic oligonucleotides with rhodamine dyes or dye networks that contain rhodamine dyes.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/776,682, filed Jan. 30, 2020, which is a divisional of U.S.application Ser. No. 15/694,279, filed Sep. 1, 2017, now abandoned,which is a divisional of U.S. application Ser. No. 14/702,499, filed May1, 2015, now U.S. Pat. No. 9,783,560, which is a divisional of U.S.non-provisional application Ser. No. 11/695,548, filed Apr. 2, 2007, nowU.S. Pat. No. 9,040,674, which claims benefit under 35 U.S.C. § 119(e)to provisional application No. 60/787,777, filed Mar. 31, 2006, whichdisclosures are herein incorporated by reference in their entirety.

2. BACKGROUND

The use of fluorescent dyes as detection labels has found widespread usein molecular biology, cell biology and molecular genetics. For example,the use of fluorescently-labeled oligonucleotides is now widespread in avariety of different assays, including polynucleotide sequencing,fluorescence in situ hybridization (FISH), hybridization assays onnucleic acid arrays, fluorescence polarization studies, and nucleic acidamplification assays, including polymerase chain amplification assayscarried out with fluorescent probes and/or primers.

Some fluorescent labels can be attached to nascent or completedoligonucleotide chains synthesized in situ using fluorescentphosphoramidite reagents. For example, fluorescein phosphoramiditereagents are available commercially (see, e.g., 2006 product catalog ofGlen Research Corporation, Sterling, Va.). In such reagents, the 3′- and6′-exocyclic oxygen atoms of the fluorescein ring are protected withpivaloyl groups to prevent side reactions. Modification of thefluorescien ring with these groups also holds the carboxylate group atthe 3-position in the closed, spiro lactone form, preventing protondonation from the carboxylate to the phosphoramidite group, which wouldconvert this phosphoramidite group into a good leaving group, leading todecomposition of the reagent. The fluorescein ring is also stable to theconditions used to oxidize the nascent oligonucleotide and to treatmentwith aqueous ammonium, the standard method by which the nucleobaseprotecting groups are removed and the synthetic oligonucleotide iscleaved from the synthesis resin.

Unfortunately, many rhodamine dyes are susceptible to chemicalmodification when treated with the reagents commonly employed to oxidizeand deprotect/cleave synthetic oligonucleotides negatively impactingtheir fluorescent properties. As a consequence, rhodamine dyes arecommonly attached to oligonucleotides following synthesis, deprotectionand cleavage from the synthesis resin. This adds additional steps andmanual labor, resulting in greater cost and inconvenience in the overallsynthesis of rhodamine-labeled oligonucleotides.

Owing to these and other limitations, there are currently only tworhodamine dyes that are commercially available as phosphoramiditereagents: tetramethyl rhodamine (“TAMRA”) and rhodamine X (“ROX”).Additional reagents that permit labeling of oligonucleotides with myriaddifferent rhodamine dyes during solid phase chemical synthesis would bedesirable.

3. SUMMARY

In one aspect, the present disclosure provides reagents useful forlabeling synthetic oligonucleotides with labels comprising rhodaminedyes that fluoresce when irradiated with incident light of anappropriate wavelength. The labels can comprise a single rhodamine dye,or they can comprise a dye network in which at least one of the dyes isa rhodamine dye. The reagents can be used to label syntheticoligonucleotides with rhodamine dye-containing labels directly duringthe step-wise synthesis of the oligonucleotides, thereby reducing themanipulation steps necessary to obtain oligonucleotides that are labeledwith rhodamine dyes. Moreover, because the labels are attached to theoligonucleotide directly during step-wise synthesis, HPLC separation ofuncoupled label from the labeled oligonucleotide, which is necessarywhen using currently available post-synthesis rhodamine labelingreagents such as rhodamine NHS esters, is unnecessary.

The reagents can be used to label an oligonucleotide at its 3′-terminus,at its 5-terminus, and/or at one or more internal positions. Theresultant label can be attached to the terminal hydroxyl(s) of theoligonucleotide, to one or more nucleobases comprising theoligonucleotide, or it can be disposed between two nucleotidescomprising the oligonucleotide chain. Thus, the reagents can take theform of non-nucleosidic synthesis reagents (see, e.g., FIGS. 3 and 5 ),nucleosidic synthesis reagents (see, e.g., FIGS. 4 and 6 ),non-nucleosidic solid supports (see, e.g., FIG. 7 ) and/or nucleosidicsolid supports (see, e.g., FIG. 8 ).

The synthesis reagents generally comprise a label moiety, a phosphateester precursor (“PEP”) group and an optional linker linking thephosphate ester precursor group to the label moiety. The phosphate esterprecursor group generally comprises a functional group that, when usedin the step-wise synthesis of oligonucleotides, ultimately yields, afteroptional deprotection and/or oxidation, the internucleotide phosphateester linkage. Several types of chemistries and functional groupssuitable for synthesizing internucleotide phosphate ester linkages areknown in the art, and include, by way of example and not limitation,phosphite triester chemistry, which utilizes phosphoramidite PEP groups(see, e.g., Letsinger et al., 1969, J. Am. Chem. Soc. 91:3350-3355;Letsinger et al., 1975, J. Am. Chem. Soc. 97:3278; Matteucci &Caruthers, 1981, J. Am. Chem. Soc. 103:3185; Beaucage & Caruthers, 1981,Tetrahedron Lett. 22:1859), phosphotriester chemistry, which utilizes2-chlorophenyl- or 2,5-dichlorophenyl-phosphate PEP groups (see, e.g.,Sproat & Gait, “Solid Phase Synthesis of Oligonucleotides by thePhosphotriester Method,” In: Oligonucleotide Synthesis, A PracticalApproach, Gait, Ed., 1984, IRL Press, pages 83-115) and H-phosphonatechemistry, which utilizes H-phosphonate PEP groups (see, e.g., Garegg etal., 1985, Chem. Scr. 25:280-282; Garegg et al., 1986, Tet. Lett.27:4051-4054; Garegg et al. 1986, Tet. Lett. 27:4055-4058; Garegg etal., 1986, Chem. Scr. 26:59-62; Froehler & Matteucci, 1986, Tet. Lett.27:469-472; Froehler et al., 1986, Nucl. Acid Res. 14:5399-5407). All ofthese various PEP groups, as well as later-discovered PEP groups, cancomprise the phosphate ester precursor group of the synthesis reagentsdescribed herein. The identity of the PEP group is not critical forsuccess, and will depend upon the desired chemistry for synthesizing thelabeled oligonucleotides. In some embodiments, the phosphate esterprecursor group comprises a phosphoramidite group.

The optional linker linking the label moiety and phosphate esterprecursor group can comprise virtually any combination of atoms orfunctional groups stable to the synthetic conditions used for thesynthesis of the labeled oligonucleotides, and can be linear, branched,or cyclic in structure, or can include combinations of linear, branchedand/or cyclic structures. The linker can be designed to have specifiedproperties, such as the ability to be cleaved under desired conditions.

The synthesis reagents may optionally further comprise one or moresynthesis handles to which nucleosides or other groups or moieties canbe attached. The synthesis handles can include protecting groups thatcan be selectively removed during the step-wise synthesis of the labeledoligonucleotide, permitting attachment of moieties to the syntheticoligonucleotide prior to cleavage from the resin, or, alternatively, thesynthesis handles can include protecting groups that are stable to theconditions used to deprotect and/or cleave the synthesizedoliognucloetide from the synthesis resin, permitting attachment ofmoieties to the synthetic labeled oligonucleotide following synthesis,deprotection and cleavage from the synthesis resin. The synthesishandles comprising a synthesis reagent that includes more than onesynthesis handle may be the same or different.

In some embodiments, the synthesis reagents comprise a single optionalsynthesis handle that comprises a protected hydroxyl of the formula—OR^(e), where R^(e) represents an acid-labile protecting group.

The synthesis handle can be linked to the label moiety, or it can beincluded in the optional linker linking the label moiety and phosphateester precursor group. Embodiments in which a synthesis handle of theformula —OR^(e) is included in the optional linker can benon-nucleosidic in nature or nucleosidic in nature, in which latter casethe linker comprises a nucleoside and the synthesis handle is providedby a hydroxyl group on the sugar moiety of a nucleoside, typically the5′-hydroxyl of the nucleoside sugar moiety. Specific, non-limitingembodiments of non-nucleosidic synthesis reagents in which the linkerincludes a synthesis handle are illustrated in FIG. 5 . Specific,non-limiting embodiments of nucleosidic synthesis reagents in which thelinker includes a synthesis handle are illustrated in FIG. 6 .

The solid support reagents generally comprise a label moiety, asynthesis handle of the formula —OR^(e) where R^(e) is as defined above,a solid support and a linker linking the label moiety and the synthesishandle to the solid support. The synthesis handle provides a group towhich nucleoside monomer reagents can be coupled. The solid supportreagents can optionally include one or more additional synthesishandles, which can be the same or different.

A wide variety of materials suitable for use as solid supports in thesolid-phase synthesis of oligonucleotides that either includeappropriate functional groups, or that can be derivatized to includeappropriate functional groups, are known in the art, and include, by wayof example and not limitation, controlled pore glass (CPG), polystyrene,and various graft co-polymers. All of these various materials aresuitable for use as the solid support in the solid support reagentsdescribed herein.

The shape of the solid support is not critical. Virtually any shape canbe utilized. For example, the solid support can be in the form ofspherical or irregularly shaped beads, cubes, rectangles, cylinders,cylindrical tubes, or even sheets. The solid support may be porous ornon-porous. In some embodiments, the solid support is a CPG orpolystyrene bead.

The linker linking the label moiety and synthesis handle to the solidsupport can comprise virtually any combination of atoms or functionalgroups stable to the synthesis conditions typically used for the solidphase synthesis of oligonucleotides, and can be linear, branched, orcyclic in structure, or can include combinations of linear, branchedand/or cyclic structures. The linker can be designed to have specifiedproperties, such as the ability to be cleaved under desired conditions.In some embodiments, the linker includes a linkage that can be cleavedunder specified conditions to release the solid support from theremainder of the reagent. For example, the linker can include linkagesthat are stable under oligonucleotides synthesis conditions and labileto the conditions used to deprotect the synthesized oligonucleotides(for example, incubation in ammonium hydroxide at 55° C. or roomtemperature). Such specifically cleavable linkages are well-known in theart, and include by way of example and not limitation, esters, carbonateesters, diisopropylsiloxy ethers, modified phosphate esters, etc.

As described above for the synthesis reagents, the linker of the solidsupport reagents can include the synthesis handle, and can benucleosidic or non-nucleosidic in nature. Specific, non-limitingembodiments of non-nucleosidic solid support reagents are illustrated inFIG. 7 . Specific, non-limiting embodiments of nucleosidic solid supportreagents are illustrated in FIG. 8 .

The label moiety of the synthesis and solid support reagents describedherein comprises a rhodamine dye. The exocyclic nitrogen atoms at the3′- and 6′-positions of the rhodamine dye are either unsubstituted ormono-substituted such they are included in a primary or secondary amine,and are further substituted with a protecting group (unprotectedrhodamine dyes having primary or secondary amine groups at their 3′- and6′-positions are referred to herein as “NH-rhodamines” and rhodaminedyes having protecting groups at their 3′- and 6′-positions are referredto herein as “N-protected NH-rhodamines”).

The protecting group can be virtually any specifically removable groupthat is stable to the synthesis conditions that will be used tosynthesize the labeled oligonucleotide, for example the phosphitetriester chemistry conditions typically used for solid-phase synthesisof oligonucleotides. It has been discovered that protecting the 3′- and6′-secondary or primary amines of NH-rhodamines with groups that formamides, such as, for example carboxamides, sulfonamides, phorphoramides,etc., permits the N-protected NH-rhodamine to exist in the closed,lactone form, thereby permitting the rhodamines to be used in thestep-wise synthesis of oligonucleotides to conveniently synthesizeoligonucleotides including rhodamine dyes without the need forpost-synthesis manipulation or purification. As demonstrated in theworking examples, phosphoramidite reagents including such N-protectedNH-rhodamines are soluble in the solvents commonly employed in thestep-wise synthesis of oligonucleotides, are stable to multiple roundsof DMT deprotection, coupling, oxidation and capping, and also totreatment with concentrated ammonium hydroxide, conditions which arecommonly used to deprotect any exocyclic amine protecting groups andcleave the synthetic oligonucleotide from the synthesis resin.

The protecting groups can be labile, and thus removable, under theconditions used to remove the nucleobase protecting groups of thesynthesized labeled oligonucleotide, or, alternatively, the protectinggroups can be stable to these conditions and labile to other conditions.In most instances, it will likely be desirable to utilize protectinggroups that are labile to the conditions used to remove the nucleobaseprotecting groups of the synthesized labeled oligonucleotide and/or tocleave the labeled synthetic oligonucleotodie from the synthesis resin.

In some embodiments, the protecting groups are acyl groups of theformula —C(O)R¹⁰, where R¹⁰ is selected from lower alkyl, methyl, —CX₃,—CHX₂, —CH₂X, —CH₂—OR^(b) and phenyl optionally mono-substituted with alower alkyl, methyl, X, —OR^(b), cyano or nitro group, where R^(b) isselected from lower alkyl, pyridyl and phenyl and each X is a halogroup, typically fluoro, chloro or bromo. In a specific, non-limitingembodiment, R¹⁰ is t-butyl or trifluoromethyl.

The label moiety may further comprise additional protected fluorophores,such that the N-protected NH-rhodamine dye is a member of a larger,energy-transfer dye network. Such energy-transfer dye networks arewell-known in the art, and include combinations of fluorophores whosespectral properties are matched, or whose relative distances to oneanother are adjusted, so that one fluorophore in the network, whenexcitated with incident irradiation of an appropriate wavelength,transfers its excitation energy to another fluorophore in the network,which in turn transfers its excitation energy to yet another fluorophorein the network, and so forth, resulting in fluorescence by the ultimateacceptor fluorophore in the network. Such networks give rise to labelshaving long Stokes shifts. In such networks, fluorophores that transfer,or donate, their excitation to another fluorophore in the network arereferred to as “donors.” Fluorophore that receive, or accept, excitationenergy from another fluorophore and fluorescein reagents thereto arereferred to as “acceptors.” In dye networks containing only two dyes,one dye typically acts as the donor and the other as the acceptor. Indye networks containing three or more different dyes, at least one dyeacts as both a donor and acceptor.

Energy transfer dye networks containing two, three, four, or even moredyes are well-known in the art (see, e.g., US 2006/057565). Any of thedyes used in these networks that can be suitably protected for use inthe solid phase synthesis oligonucleotides can be included in the labelmoieties described herein.

In some embodiments of the synthesis and solid support reagentsdescribed herein, the label moiety further comprises a suitablyprotected donor for the N-protected NH-rhodamine. When deprotected, suchdonors transfer their excitation energy to the NH-rhodamine such thatthe NH-rhodamine emits fluorescence upon excitation of the donor.

In some embodiments of the synthesis and solid support reagentsdescribed herein, the label moiety further comprises a suitablyprotected acceptor for the N-protected NH-rhodamine. When deprotected,such acceptors accept excitation energy from the donor NH-rhodamine suchthat the acceptor fluoresces upon excitation of the donor NH-rhodamine.

The identities of the donor or acceptor will depend upon the identity ofthe NH-rhodamine comprising the label moiety. Examples of fluorophorescapable of acting as a donor for a wide variety of rhodamine dyes arewell-known in the art. Non-limiting examples of such donors includexanthene dyes (such as, for example,) fluoresceins, rhodamines andrhodols), pyrene dyes, coumarin dyes (for example hydroxy and aminocoumarins), cyanine dyes, phthalocyanine dyes, and lanthenide complexes.Examples of fluorophores capable of acting as acceptors for rhodaminedyes are also well-known in the art. Non-limiting examples of suchacceptors include rhodamines dyes and cyanines dyes. Any of these dyesthat can be suitably protected for use under the conditions used tosynthesize oligonucleotides, such as the phosphite triester chemistryconditions typically used for the solid-phase synthesis ofoligonucleotides, can be used as the donor or acceptor in the synthesisand solid support reagents described herein.

The mechanism by which energy is transferred from a donor to theacceptor is not critical. All that is necessary for such donor-acceptorpairs to be operable is that the acceptor fluoresce in response toexcitation of the donor.

The label moiety may also include acceptors that are non-fluorescent.Such non-fluorescent acceptors can be used to quench, either in whole orin part, the fluorescence of the NH-rhodamine or other fluorescentdye(s) comprising the label moiety. Examples of such non-fluorescentmoieties that can act as quenchers for rhodamine dyes such as theNH-rhodamines described herein include, but are not limited to, dabcyl,the various non-fluorescent quenchers described in WO 01/86001and thevarious non-fluorescent quenchers described in US 2005/0164225, thedisclosures of which are incorporated herein by reference.

In some embodiments, the label moiety further comprises a donor dye thatin turn comprises an N-protected NH-rhodamine dye as described herein ora fluorescein dye in which the exocylic 3′- and 6′-oxygen atoms areprotected with protecting groups that are stable to oligonucleotidesynthesis conditions, such as the phosphite triester chemistryconditions typically used for the solid phase synthesis ofoligonuclotides, and labile to the conditions used to deprotect thesynthesized oligonucleotide. Suitable protecting groups are well-knownin the art and include, by way of example and not limitation, acylgroups carbonates and carbamates. Fluorescein dyes including protectinggroups at the 3′- and 6′-exocyclic oxygen atoms are referred to hereinas “O-protected fluoresceins.” In a specific, non-limiting embodiment,the protecting groups on the O-protected fluorescein are acyl groups.

The N-protected NH-rhodamine or O-protected fluorescein donor dye andthe N-protected NH-rhodamine acceptor dye can be linked to one anotherin a variety of orientations, either directly or with the aid of alinker. In some embodiments, the donor is linked to the 2′-, 2″-, 4′-,5′-, 7′, 7″-, 5- or 6-position of the N-protected NH-rhodamine acceptorvia its 2′-, 2″-, 4′-, 5′-, 7′-, 7″-, 5- or 6-position, optionally withthe aid of a linker. In some embodiments, the donor and acceptor arelinked to one another in a head-to-head, head-to-tail, tail-to-tail orside-to-side orientation, as will be described in more detail in a latersection.

The optional linker linking the dyes may comprise virtually anycombination of atoms and/or functional groups stable to oligonucleotidesynthesis conditions, such as the phosphite triester chemistryconditions typically used for the solid phase synthesis ofoligonucleotides. Combinations of atoms and/or functional groups can beselected to tailor the properties and/or length of the linker asdesired. A variety of linkers useful for linking fluorescein andrhodamine dyes to one another in the context of energy transfer dyenetworks are known in the art (see, e.g., US 2006/057565, U.S. Pat. Nos.7,015,000, 6,627,748, 6,544,744, 6,177,247, 6,150,107, 6,028,190,5,958,180, 5,869,255, 5,853,992, 5,814,454, 5,804,386, 5,728,528,5,707,804 and 5,688,648, the disclosures of which are incorporatedherein by reference.) All of these linkers can be used to linkO-protected fluoresceins to N-protected NH-rhodamines in the reagentsdescribed herein. In some embodiments, the linker is rigid in nature andis about 8 to 16 Å in length.

The label moiety, phosphate ester precursor group and optional synthesishandle of the synthesis reagents, and the label moiety, solid supportand synthesis handle of the solid support reagents can be linked to oneanother in any fashion or orientation that does not interfere with theabilities of the various groups and moieties to carry out theirrespective functions. The label moiety is typically linked to one of theother moieties or groups comprising the particular reagent via a linkerlinked to a functional group on one of the dyes comprising the labelmoiety, or, alternatively, to a linker linking two or more dyes of a dyenetwork. In embodiments in which the label moiety includes only a singleN-protected NH-rhodamine fluorophore, the label moiety can be linked tothe reagent via any position of the NH-rhodamine ring that does notinterfere with the ability of the NH-rhodamine ring to exist in theclosed, spiro lactone form. Suitable positions include, but are notlimited to, carbon atoms that are adjacent to an exocylic nitrogen atom,or atoms on the phenyl moiety, such as, for example, the 2′-, 2″-, 4′-,5′-, 7′-, 7″-, 5- or 6-position of the rhodamine dye. In someembodiments, the label moiety is linked to a group or moiety of thereagent via the 5- or 6-position of the N-protected NH-rhodamine dye.

In embodiments in which the label moiety comprises a dye network, thelabel moiety may be linked to any group or moiety of the reagent at anyposition on any one of the dyes comprising the network that does notinterfere with the desired function of the reagent, or to a linkerlinking two or more dyes of the network. In some specific embodiments inwhich the label moiety comprises an O-protected fluorescein orN-protected NH-rhodamine donor in addition to the N-protectedNH-rhodamine acceptor, the label moiety can be linked to a group ormoiety of the reagent at any available position on the donor oracceptor, such as, for example, the 2′-, 2″-, 4′-, 5′-, 7′-, 7″-, 5- or6-position of the donor or acceptor. In some embodiments, the labelmoiety is linked to a group or moiety of the regent via the 5-or6-position of the donor or acceptor.

In some embodiments, the label moiety is linked to a group or moiety ofthe reagent via the linker linking the donor and N-protectedNH-rhodamine acceptor.

In other aspects, the disclosure provides intermediate molecules usefulfor synthesizing the reagents described herein, methods of making thereagents described herein, compounds labeled with the reagents describedherein, such as, for example, labled oligo or polynucleotides, andmethods of using the labeled compounds in a variety of contexts. All ofthe various aspects of the disclosure are described in more detailbelow.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B provide exemplary embodiments of parent NH-rhodaminedyes that can be incorporated into the reagents described herein;

FIG. 1C provides exemplary embodiments of parent fluorescein dyes thatcan act as donors for NH-rhodamines, and that can be incorporated intothe reagents described herein in embodiments in which the label moietycomprises a dye network;

FIG. 2 provides exemplary linkers that can be used to link the variousdifferent moieties comprising the reagents described herein to oneanother;

FIG. 3 provides exemplary embodiments of non-nucleosidic synthesisreagents that do not include synthesis handles;

FIG. 4 provides exemplary embodiments of nucleosidic synthesis reagentsthat do not include synthesis handles;

FIG. 5 provides exemplary embodiments of non-nucleosidic synthesisreagents that include a synthesis handle;

FIG. 6 provides exemplary embodiments of nucleosidic synthesis reagentsthat include synthesis handles;

FIG. 7 provides exemplary embodiments of non-nucleosidic solid supportreagents;

FIG. 8 provides exemplary embodiments of nucleosidic solid supportreagents;

FIG. 9 illustrates the use of a specific embodiment of a synthesisreagent to synthesize an oligonucleotide labled at its 5′-hydroxyl withan NH-rhodamine dye;

FIG. 10 illustrates the use of a specific embodiment of a synthesisreagent to synthesize an oligonucleotide labeled at its 3-hydroxyl withan energy-transfer dye;

FIG. 11A illustrates the use of a specific embodiment of a synthesisreagent to synthesize in situ an oligonucleotide labeled at its5′-hydroxyl with an energy-transfer dye; and

FIG. 11B illustrates the use of a linker phosphoramidite and a specificembodiment of a synthesis reagent to synthesize in situ anoligonucleotide labeled at its 5^(,)-terminus with an energy transferdye.

5. DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not intended to be restrictive of the compositions and methodsdescribed herein. In this disclosure, the use of “or” means “and/or”unless stated otherwise. Similarly, the expressions “comprise,”“comprises,” “comprising,” “include,” “includes” and “including” are notintended to be limiting.

5.1 Definitions

As used herein, the following terms and phrases are intended to have thefollowing meanings:

Alkyl,” by itself or as part of another substituent, refers to asaturated or unsaturated branched, straight-chain or cyclic, monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. As used herein,“lower alkyl” means (C1-C8) alkyl.

“Alkanyl,” by itself or as part of another substituent, refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. Asused herein, “lower alkanyl” means (C1-C8) alkanyl.

“Alkenyl,” by itself or as part of another substituent refers, to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. As used herein, “lower alkenyl” means (C2-C8) alkenyl.

“Alkynyl,” by itself or as part of another substituent, refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. As used herein, “loweralkynyl” means (C2-C8) alkynyl.

“Alkyldiyl,” by itself or as part of another substituent, refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (i.e., C1-C6means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typicalalkyldiyl groups include, but are not limited to, methandiyl; ethyldiylssuch as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies are on the samecarbon atom, the nomenclature “alkylidene” is used. In some embodiments,the alkyldiyl group is (C1-C8) alkyldiyl. Specific embodiments includesaturated acyclic alkanyldiyl groups in which the radical centers are atthe terminal carbons, e.g., methandiyl (methano); ethan-1,2-diyl(ethano); propan-1,3-diyl (propano); butan-1,4-diyl (butano); and thelike (also referred to as alkylenos, defined infra). As used herein,“lower alkyldiyl” means (C1-C8) alkyldiyl.

“Alkylene,” by itself or as part of another substituent, refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of two terminal carbon atoms of straight-chainor branched parent alkane, alkene or alkyne, or by the removal of onehydrogen atom from each of two different ring atoms of a parentcycloalkyl. The locant of a double bond or triple bond, if present, in aparticular alkylene is indicated in square brackets. Typical alkylenegroups include, but are not limited to, methylene (methano); ethylenessuch as ethano, etheno, ethyno; propylenes such as propano, prop[1]eno,propa[1,2]dieno, prop[1]yno, etc.; butylenes such as butano, but[1]eno,but[2]eno, buta[1,3]dieno, but[1]yno, but[2]yno, buta[1,3]diyno, etc.;and the like. Where specific levels of saturation are intended, thenomenclature alkano, alkeno and/or alkyno is used. In some embodiments,the alkylene group is (C1-C8) or (C1-C3) alkylene. Specific embodimentsinclude straight-chain saturated alkano groups, e.g., methano, ethano,propano, butano, and the like. As used herein, “lower alkylene” means(C1-C8) alkylene.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” Heteroalkynyl,”Heteroalkyldiyl” and “Heteroalkylene,” by themselves or as part ofanother substituent, refer to alkyl, alkanyl, alkenyl, alkynyl,alkyldiyl and alkylene groups, respectively, in which one or more of thecarbon atoms are each independently replaced with the same or differentheteroatoms or heteroatomic groups. Typical heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —SO₂—, —S(O)NR′—,—SO₂NR′—, and the like, including combinations thereof, where R′ ishydrogen or a substitutents, such as, for example, (C1-C8) alkyl,(C6-C14) aryl or (C7-C20) arylalkyl.

“Cycloalkyl” and “Heterocycloalkyl,” by themselves or as part of anothersubstituent, refer to cyclic versions of “alkyl” and “heteroalkyl”groups, respectively. For heteroalkyl groups, a heteroatom can occupythe position that is attached to the remainder of the molecule. Typicalcycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. Typical heterocycloalkyl groups include, butare not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-l-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), and the like.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated 7C electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene,tetrahydronaphthalene, etc. Typical parent aromatic ring systemsinclude, but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,tetrahydronaphthalene, triphenylene, trinaphthalene, and the like.

“Aryl,” by itself or as part of another substituent, refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C6-C14 means from 6 to 14 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene, and the like, as well as the various hydroisomers thereof Specific exemplary aryls include phenyl and naphthyl.

“Arylalkyl,” by itself or as part of another substituent, refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, in some embodiments a terminal or sp^(a) carbon atom, isreplaced with an aryl group. Typical arylalkyl groups include, but arenot limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl,naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl,naphthobenzyl, 2-naphthophenylethan-1-yl and the like. Where alkylmoieties having a specified degree of saturation are intended, thenomenclature arylalkanyl, arylalkenyl and/or arylalkynyl is used. When adefined number of carbon atoms are stated, for example, (C7-C20)arylalkyl, the number refers to the total number of carbon atomscomprising the arylalkyl group.

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms are each independently replacedwith the same or different heteroatoms or heteroatomic groups. Typicalheteroatoms or heteroatomic groups to replace the carbon atoms include,but are not limited to, N, NH, P, O, S, S(O), SO₂, Si, etc. Specificallyincluded within the definition of “parent heteroaromatic ring systems”are fused ring systems in which one or more of the rings are aromaticand one or more of the rings are saturated or unsaturated, such as, forexample, benzodioxan, benzofuran, chromane, chromene, indole, indoline,xanthene, etc. Also included in the definition of “parent heteroaromaticring system” are those recognized rings that include commonsubstituents, such as, for example, benzopyrone and1-methyl-1,2,3,4-tetrazole. Typical parent heteroaromatic ring systemsinclude, but are not limited to, acridine, benzimidazole, benzisoxazole,benzodioxan, benzodioxole, benzofuran, benzopyrone, benzothiadiazole,benzothiazole, benzotriazole, benzoxaxine, benzoxazole, benzoxazoline,carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

“Heteroaryl,” by itself or as part of another substituent, refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof.

“Heteroarylalkyl,” by itself or as part of another substituent, refersto an acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, in some embodiments a terminal or sp³ carbon atom, isreplaced with a heteroaryl group. Where alkyl moieties having aspecified degree of saturation are intended, the nomenclatureheteroarylalkanyl, heteroarylalkenyl and/or heteroarylalkynyl is used.When a defined number of atoms are stated, for example, 6-20-memberedhetoerarylalkyl, the number refers to the total number of atomscomprising the arylalkyl group.

“Haloalkyl,” by itself or as part of another substituent, refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C1-C2) haloalkyl” includes fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As non-limiting specific examples, “alkyloxy” and/or“alkoxy” refer to a group of the formula —OR″, “alkylamine” refers to agroup of the formula —NHR″ and “dialkylamine” refers to a group of theformula —NR″R″, where each R″ is an alkyl.

5.2 Exemplary Embodiments

The present disclosure provides reagents that can be used to chemicallysynthesize oligonucleotides bearing label moieties that compriserhodamine dyes. Traditionally, it has been difficult to chemicallysynthesize rhodamine-labeled oligonucleotides owing, in part, to thelack of availability of rhodamine-containing synthesis reagents that arestable to the synthesis and/or deprotection conditions commonly employedin the step-wise chemical synthesis of oligonucleotides. It has now beendiscovered that protecting the exocyclic amine groups of NH-rhodaminedyes with base-labile protecting groups, such as acetyl groups, providesN-protected NH-rhodamine dyes that are stable to the chemical synthesisand deprotection conditions commonly employed in the solid-phasesynthesis of oligonucleotides. As a consequence, the N-protectedNH-rhodamines can be incorporated into reagents that can be used tosynthesize oligonucleotides labeled with label moieties that compriserhodamine dyes, thereby obviating the need to attach the labelspost-synthesis. Because the labels are attached during synthesis, theresultant labeled oligonucleotide can be purified for use without theuse of HPLC.

The reagents take advantage of various features of reagents andchemistries that are well-known for the step-wise solid phase synthesisof oligonucleotides, and can be in the form of synthesis reagents thatare coupled to a hydroxyl group during the step-wise solid phasesynthesis of an oligonucleotide chain, or in the form of solid supportreagents to which nucleoside monomer reagents, such as nucleosidephosphoramidite reagents, and/or optionally other reagents, are coupledin a step-wise fashion to yield a synthetic oligonucleotide.

The synthesis and solid support reagents can be nucleosidic in nature inthat they can include a nucleoside moiety, or they can benon-nucleosidic in nature.

All of the reagents described herein include a label moiety thatcomprises an N-protected NH-rhodamine dye or moiety. The N-protectedNH-rhodamine dye can be the only dye comprising the label moiety or,alternatively, it can be one of two or more dyes comprising a larger dyenetwork. The solid support reagents additionally include a solid supportand one or more synthesis handles to which additional groups can becoupled. The synthesis reagents additionally include a phosphate esterprecursor group useful for coupling the reagent to a primary hydroxylgroup, and may optionally include one or more synthesis handles. Thevarious moieties and groups comprising the reagents can be linkedtogether in any fashion and/or orientation that permits them to carryout their respective functions. They can be linked to one anotherthrough linking groups included on the moieties, or they can be linkedto one another with the aid of linkers.

The various moieties, groups and linkers comprising the reagentsdescribed herein are described in more detail below.

5.3 Linkers and Linking Groups

The various groups and moieties comprising the reagents described hereinare typically connected to one another with linkers. The identity of anyparticular linker will depend, in part, upon the identities of themoieties being linked to one another. In general, the linkers include aspacing moiety that can comprise virtually any combination of atoms orfunctional groups stable to the synthetic conditions used for thesynthesis of labeled oligonucleotides, such as the conditions commonlyused to synthesize oligonucleotides by the phosphite triester method,and can be linear, branched, or cyclic in structure, or can includecombinations of linear, branched and/or cyclic structures. The spacingmoiety can be monomeric in nature, or it can be or include regions thatare polymeric in nature. The spacing moiety can be designed to havespecified properties, such as the ability to be cleaved under specifiedconditions, or specified degrees of rigidity, flexibility,hydrophobicity and/or hydrophilicity.

As will be described in more detail below, many embodiments of thereagents described herein are synthesized by condensing synthons to oneanother in specified fashions to yield the desired reagents. Eachsynthon typically includes one or more linking groups suitable forforming the desired linkages. Generally, the linking group comprises afunctional group F^(y) that is capable of reacting with, or that iscapable of being activated so as to be able to react with, anotherfunctional group F^(z) to yield a covalent linkage Y-Z, where Yrepresents the portion of the linkage contributed by F^(y) and Z theportion contributed by F^(z). Such groups F^(y) and F^(z) are referredto herein as “complementary functional groups.”

Pairs of complementary functional groups capable of forming covalentlinkages with one another are well-known in the art. In someembodiments, one of F^(y) or F^(z) comprises a nucleophilic group andthe other one of F^(y) or F^(z) comprises an electrophilic group.Complementary nucleophilic and electrophilic groups useful for forminglinkages (or precursors thereof that are or that can be suitablyactivated so as to form linkages) that are stable to a variety ofsynthesis and other conditions are well-known in the art. Examples ofsuitable complementary nucleophilic and electrophilic groups that can beused to effect linkages in the various reagents described herein, aswell as the resultant linkages formed therefrom, are provided in Table1, below:

TABLE 1 Resultant Covalent Electrophilic Group Nucleophilic GroupLinkage activated esters* amines/anilines carboxamides acyl azides**amines/anilines carboxamides acyl halides amines/anilines carboxamidesacyl halides alcohols/phenols esters acyl nitriles alcohols/phenolsesters acyl nitriles amines/anilines carboxamides aldehydesamines/anilines imines aldehydes or ketones hydrazines hydrazonesaldehydes or ketones hydroxylamines oximes Alkyl halides amines/anilinesalkyl amines Alkyl halides carboxylic acids esters Alkyl halides thiolsthioethers Alkyl halides alcohols/phenols ethers Alkyl sulfonates thiolsthioethers Alkyl sulfonates carboxylic acids esters Alkyl sulfonatesalcohols/phenols esters anhydrides alcohols/phenols esters anhydridesamines/anilines caroboxamides aryl halides thiols thiophenols arylhalides amines aryl amines aziridines thiols thioethers boronatesglycols boronate esters carboxylic acids amines/anilines carboxamidescarboxylic acids alcohols esters carboxylic acids hydrazines hydrazidescarbodiimides carboxylic acids N-acylureas or anhydrides diazoalkanescarboxylic acids esters epoxides thiols thioethers haloacetamides thiolsthioethers halotriazines amines/anilines aminotriazines halotriazinesalcohols/phenols triazinyl ethers imido esters amines/anilines amidinesisocyanates amines/anilines ureas isocyanates alcohols/phenols urethanesisothiocyanates amines/anilines thioureas maleimides Thiols thioethersphosphoramidites Alcohols phosphate esters silyl halides Alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate estersThiols thioethers sulfonate esters carboxylic acids esters sulfonateesters Alcohols esters sulfonyl halides amines/anilines sulfonamidessulfonyl halides phenols/alcohols sulfonate esters Diazonium salt arylazo *Activated esters, as understood in the art, generally have theformula —C(O)Ω, where Ω is a good leaving group (e.g., oxysuccinimidyl,oxysulfosuccinimidyl, 1-oxybenzotriazolyl, etc.). **Acyl azides canrearrange to isocyanates.

Thus, linker synthons can generally be described by the formulaLG-Sp-LG, where each LG represents, independently of the other, alinking group, and Sp represents the spacing moiety. In someembodiments, linker synthons can be described by the formulaF^(z)-Sp-F^(z), where each F^(z) represents, independently of the other,one member of a pair of complementary nucleophilic or electrophilicfunctional groups as described above. In specific embodiments, eachF^(z) is, independently of the other, selected from the groups listed inTable 1, supra. Linker synthons of this type form linker moieties of theformula —Z-Sp-Z—, where each Z represents, independently of the other, aportion of a linkage as described above.

Specific linkers suitable for linking specified groups and moieties toone another in the reagents described herein will be discussed in moredetail in connection with exemplary embodiments of the reagents.Non-limiting exemplary embodiments of linkers that can be used to linkthe various groups and moieties comprising the reagents described hereinto one another are illustrated in FIG. 2 . In FIG. 2 , Z¹ and Z² eachrepresent, independently of one another, a portion of a linkagecontributed by a functional group F^(z), as previously described, and Kis selected from —CH— and —N—. In some specific embodiments of thelinkers illustrated in FIG. 2 , one of Z¹ or Z² is —NH— and the other isselected from —O—, —C(O)— and —S(O)₂—.

5.4 Label Moiety

All of the reagents described herein include a label moiety thatcomprises an NH-rhodamine dye that is protected at the exocyclic aminegroups with a protecting group having specified properties. Generally,rhodamine dyes are characterized by four main features: (1) a parentxanthene ring; (2) an exocyclic amine substituent; (3) an exocyclicimminium substituent; and (4) a phenyl group substituted at the orthoposition with a carboxyl group. The exocyclic amine and/or imminiumgroups are typically positioned at the C3 and C6 carbon atoms of theparent xanthene ring, although “extended” rhodamines in which the parentxanthene ring comprises a benzo group fused to the C3 and C4 carbonsand/or the C5 and C6 carbons are also known. In these extendedrhodamines, the characteristic exocyclic amine and imminium groups arepositioned at the corresponding positions of the extended xanthene ring.

The carboxyl-substituted phenyl group is attached to the C9 carbon ofthe parent xanthene ring. As a consequence of the ortho carboxylsubstituent, rhodamine dyes can exist in two different forms:(1) theopen, acid form; and (2) the closed, lactone form. While not intendingto be bound by any theory of operation, because NMR spectra of exemplaryN-protected NH-rhodamine dyes described herein are consistent with theclosed spiro lactone form of the dye, it is believed that theN-protected NH-rhodamine dyes comprising the label moiety of thereagents described herein are in the closed, spiro lactone form. Thus,the various rhodamines, as well as their unprotected counterparts, areillustrated herein in their closed, spiro lactone form. However, it isto be noted that this is for convenience only and is not intended tolimit the various reagents described herein to the lactone form of thedyes.

In the closed, spiro lactone form, the A and C rings of the parentxanthene ring are aromatic, and both the C3′ and C6′ substituents areamines. The exocyclic amine groups of the rhodamine dyes included in thelabel moieties described herein are either unsubstituted ormono-substituted such that these amine groups are primary or secondaryamines. Such rhodamine dyes are referred to herein as “NH-rhodamines.”Thus, as used herein, an “NH-rhodamine” generally comprises one of thefollowing parent NH-rhodamine ring structures:

In the parent NH-rhodamine rings depicted above, the various carbonatoms are numbered using an arbitrary numbering convention adopted froma numbering convention commonly used for the closed, spiro lactone formof rhodamine dyes. This numbering system is being used for convenienceonly, and is not intended to be limiting in any way.

In the parent NH-rhodamines rings of structural formula (Ia), (Ib) and(Ic), R^(3′) and R^(6′) represent hydrogen or substituent groupssubstituting the exocyclic amines. The R^(3′) and/or R^(6′) substituentscan be the same or different, and can comprise groups such assubstituted or unsubstituted alkyl, aryl or arylalkyl groups.Alternatively, the R^(3′) and/or R^(6′) groups can comprise substituentsthat are bridged to an adjacent carbon atom such that the illustratednitrogen atom is included in a ring that contains 5- or 6-ring atoms.The ring may be saturated or unsaturated, and one or more of the ringatoms can be substituted. When the ring atom(s) are substituted, thesubstituents are typically, independently of one another, selected fromlower alkyl, C6-C10 aryl and C7-C16 arylalkyl groups. Alternatively, twoadjacent ring atoms may be included in an aryl bridge, such as a benzoor naphtho group. Non-limiting exemplary embodiments of rhodamine dyesthat include a parent NH-rhodamine ring according to structural formula(Ia) in which the R^(3′) and/or R^(6′) groups are hydrogen or loweralkyl groups or are included in optionally substituted rings withadjacent carbon atoms are illustrated in FIG. 1A. Non-limiting exemplaryembodiments of rhodamine dyes that include a parent NH-rhodamine ringaccording to structural formula (Ic) in which the R^(3′) and R^(6′)groups are hydrogen or lower alkyl groups or are included in optionallysubstituted rings with adjacent carbon atoms are illustrated in FIG. 1B.

One or more of the carbon atoms at positions C1′, C2′, C2″, C4′, C4″,C5′, C5″, C7′, C7″ and C8′ of the parent NH-rhodamine rings according tostructural formulae (Ia), (Ib) and (Ic) can be, independently of oneanother, substituted with substituent groups. Groups useful forsubstituting rhodamine dyes at these positions are well known in theart, and are described, for example, in U.S. Pat. Nos. 4,622,400,5,750,409, 5,847,162, 6,017,712, 6,080,852, 6,184,379 and 6,248,884, thedisclosures of which are incorporated herein by reference. All of thesesubstituent groups can be used to substitute the parent NH-rhodaminerings described herein.

In some embodiments, the substituent groups are, independently of oneanother, selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl,5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, —R^(b) and—(CH₂)_(x)—R^(b), where x is an intger ranging from 1 to 10 and R^(b) isselected from —X, —OH, —SH, —SR^(a), —NH₂, —NHR^(a), —NR^(c)R^(c),—N⁺R^(c)R^(c)R^(c), perhalo lower alkyl, trihalomethyl, trifluoromethyl,—B(OH)₃, —B(OR^(a))₃, —B(OH)O⁻, —B(OR^(a))₂O⁻, —B(OH)(O⁻)₂,—B(OR^(a))(O⁻)₂, —P(OH)₂, —P(OH)O⁻, —P(OR^(a))₂, —P(OR^(a))O⁻,—P(O)(OH)₂, —P(O)(OH)O⁻, —P(O)(O⁻)₂, —P(O)(OR^(a))₂, —P(O)(OR^(a))O⁻,—P(O)(OH)(OR^(a)), —OP(OH)₂, —OP(OH)O⁻, —OP(OR^(a))₂, —OP(OR^(a))O⁻,—OP(O)(OH)₂, —OP(O)(OH)O⁻, —OP(O)(O⁻)₂, —OP(O)(OR^(a))₂,—OP(O)(OR^(a))O⁻, —OP(O)(OR^(a))(OH), —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R^(a),—C(O)H, —C(O)R^(a), —C(S)X, —C(O)O⁻, —C(O)OH, —C(O)NH₂, —C(O)NHR^(a),—C(O)NR^(c)R^(c), —C(S)NH₂, —C(O)NHR^(a), —C(O)NR^(c)R^(c), —C(NH)NH₂,—C(NH)NHR^(a), and —C(NH)NR^(c)R^(c), where X is a halo (preferablyfluoro or chloro), each R^(a) is, independently of the others, selectedfrom lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 memberedheteroaryl and 6-20 membered heteroarylalkyl, and each R^(c) is,independently of the others, an R^(a), or, alternatively, two R^(c)bonded to the same nitrogen atom may be taken together with thatnitrogen atom to form a 5- to 8-membered saturated or unsaturated ringthat may optionally include one or more of the same or different ringheteroatoms, which are typically selected from O, N and S.

Alternatively, the C1′ and C2′ substituents, the C7′ and C8′substituents, the C5′ and C5″ substituents and/or the C4′ and C4″substituents can be taken together to form substituted or unsubstitutedaryl bridges, such as benzo bridges, with the proviso that the C1′ andC2′ substituents, and C7′ and C8′ substituents are not simultaneouslyincluded in an aryl bridge.

In general, the groups used to substitute the C1′, C2′, C2″, C4′, C4″,C5′, C5″, C7′, C7″ and C8′ carbons should not promote quenching of therhodamine dye, although in some embodiments quenching substituents maybe desirable. Substituents that tend to quench rhodamine dyes includecarbonyl, carboxylate, heavy metals, nitro, bromo and iodo. Phenylgroups positioned at R³′ and/or R⁶′ also tend to cause quenching.

The carbon atoms at positions C4, C5, C6 and C7 of the parentNH-rhodamine rings of structural formulae (Ia), (Ib) and (Ic) can also,independently of one another, include optional substituents. Thesesubstituents can be selected from the various substituents describedabove. In some embodiments, the carbon atoms at positions C4 and C7 aresubstituted with chloro groups such that the parent NH-rhodamine dye isan NH-4,7-dichlororhodamine dye.

A vast number of rhodamine dyes that include parent NH-rhodamine ringsaccording to structural formulae (Ia), (Ib) and (Ic) that can beincluded in the label moiety of the reagents described herein are knownin the art, and are described, for example, in U.S. Pat. No. 6,248,884;6,111,116; 6,080,852; 6,051,719; 6,025,505; 6,017,712; 5,936,087;5,847,162; 5,840,999; 5,750,409; 5,366,860; 5,231,191; 5,227,487; WO97/36960; WO 99/27020; Lee et al., 1992, Nucl. Acids Res. 20:2471-2483;Arden-Jacob, “Neue Lanwellige Xanthen-Farbstoffe für Fluoreszenzsondenand Farbstoff Lauer, Springer-Verlag, Germany, 1993; Sauer et al., 1995,Fluorescence 5:247-261; Lee et al., 1997, Nucl. Acids Res. 25:2816-2822;and Rosenblum et al., 1997, Nucl. Acids Res. 25:4500-4504, thedisclosures of which are incorporated herein by reference. Any of thedyes described in these references in which the exocyclic amines areprimary or secondary amines as described herein, or 4,7-dichloroanalogues of such NH-rhodamine dyes, can be included in the label moietyof the reagents described herein.

When included in a label moiety, the exocyclic amines of the parentNH-rhodamine ring are protected with protecting groups having specifiedproperties. Such protected NH-rhodamines are referred to herein as“N-protected NH-rhodamines.” The N-protected NH-rhodamines thatcorrespond to the parent NH-rhodamine rings of structural formulae (Ia),(Ib) and (Ic) and are illustrated below as structural formulae (IIa),(IIb) and (IIc):

In structural formulae (IIa), (IIb) and (IIc), R′ is hydrogen or R^(3′)and R″ is hydrogen or R^(6′), where R^(3′) and R^(6′) are as defined forstructural formulae (Ia), (Ib) and (Ic), supra, and R⁹ represents aprotecting group. The N-protected NH-rhodamines can include substituentsat one or more of positions C1′, C2′, C2″, C4′, C4″, C5′, C5″, C7′, C7″,C8′, C4, C5, C6, and C7, as previously described in connection with theparent NH-rhodamine rings according to structural formulae (Ia), (Ib)and (Ic).

Since the reagents described herein will be used to chemicallysynthesize labeled oligonucleotides, protecting groups R⁹ that arestable to the organic synthesis conditions used to synthesizeoligonucleotides should be used. As mentioned above, protecting groupsR⁹ that protect the amine in the form of an amide, for example, acarboxamide, a sufonamide or a phosphoramide, should be selected, asprotecting the exocyclic amines in this matter is believed to “lock” theprotected NH-rhodamine in the closed, lactone, form, contributing to thestability of the reagents described herein. Although not required, it isconvenient to utilize protecting groups R⁹ that are labile under theconditions used to remove the groups protecting the exocyclic amines ofthe nucleobases of the synthetic oligonucleotide, so that all protectinggroups can be removed in a single step.

The conditions used to synthesize and deprotect syntheticoligonucleotides are well-known in the art, and are described, forexample, in Current Protocols in Nucleic Acid Chemistry, Vol. I,Beancage et al., Eds., John Wiley & Sons, 2002, the disclosure of whichare incorporated herein by reference. Briefly, synthesis methods thatemploy phosphoramidite reagents involve multiple rounds of (i) DMTdeprotection to reveal a free hydroxyl, which can be effected bytreatment with 2.5% or 3% di- or tri-chloroacetic acid indichloromethane; (ii) coupling of nucleoside or other phosphoramiditereagents to the free hydroxyl, which can be carried out in acetonitrilecontaining 0.45 M or 0.5 M tetrazole; (iii) oxidation, which can becarried out by treatment with I₂/2,6-lutidine/H₂O; and capping, whichcan be carried out by treatment with 6.5% acetic anhydride intetrahydrofuran (THF) followed by treatment with 10% 1-methylimidazole(NMI) in THF.

Other conditions for carrying out the various steps in the synthesis arealso known and used. For example, phosphoramidite coupling can becarried out in acetonitrile containing 0.25 M 5-ethylthio-1H-tetrazole,0.25 M4,5-dicyanoimidazole (DCI) or 0.25 M 5-benzylthio-1H-tetrazole(BTT). Oxidation an be carried out in 0.1 M, 0.05 M or 0.02 M I₂ inTHF/H₂O/pyridine (7:2:1). Capping can be carried out by treatment withTHF/lutidine/acetic anhydride followed by treatment with 16% NMI in THF;by treatment with 6.5% DMAP in THF followed by treatment with 10% Melmin THF; or by treatment with 10% Melm in THF followed by treatment with16% Melm in THF.

Removal of any protecting groups and cleavage from the synthesis reagentis typically effected by treatment with concentrated ammonium hydroxideat 60° C. for 1-12 hr, although nucleoside phosphoramidite reagentsprotected with groups that can be removed under milder conditions, suchas by treatment with concentrated ammonium hydroxide at room temperaturefor 4-17 hrs or treatment with 0.05 M potassium carbonate in methanol,or treatment with 25% t-butylamine in H₂O/EtOH, are also known and used.

Skilled artisans will be readily able to select protecting groups havingproperties suitable for use under specific synthesis and deprotectionand/or cleavage conditions. A wide variety of amine protecting groupsare taught, for example in, Greene & Wuts, “Protective Groups In OrganicChemistry,” 3d Edition, John Wiley & Sons, 1999 (hereinafter “Green &Wuts”) at for example, pages 309-405. Skilled artisans can readilyselect protecting groups R⁹ having suitable properties from amongstthose taught in Green & Wuts.

In some embodiments, the protecting groups R⁹ are acyl groups of theformula —C(O)R¹⁰, where R¹⁰ is selected from hydrogen, lower alkyl,methyl, —CX₃, —CHX₂, —CH₂X, —CH₂—OR^(d) and phenyl optionallymono-substituted with a lower alkyl, methyl, —X, —OR^(d), cyano or nitrogroup, where R^(d) is selected from lower alkyl, phenyl and pyridyl, andeach X is a halo group, typically fluoro, or chloro. In someembodiments, R^(I)° is methyl. In some embodiments, R¹⁰ istrifluoromethyl.

Acyl protecting groups such as those defined by —C(O)R¹⁰ can be removedunder a variety of basic conditions, including the mild conditions usedto remove protecting groups from oligos synthesized with “base labile”phosphoramidite reagents, as are well-known in the art. Exemplaryconditions that can be used are specified above.

As will be described in more detail in later sections, the N-protectedNH-rhodamine moiety comprising the label moiety may be linked to othergroups or moieties. For example, the N-protected NH-rhodamine may belinked to another dye comprising the label moiety, to a phosphate esterprecursor group, to a linker, to a synthesis handle, to a quenchingmoiety, to a moiety that functions to stabilize base-pairinginteractions (such as, for example an intercalating dye or aminor-groove-binding molecule), or to other moieties. Such linkages aretypically effected via linking groups LG (described above in connectionwith the linkers) attached to the N-protected NH-rhodamine synthons usedto synthesize the reagents.

The linking group LG can be attached to any available carbon atom of theN-protected NH-rhodamine synthon, or to a substituent group attached toone of these carbon atoms. The positions of the linking groups maydepend, in part, on the group or moiety to which the N-protectedNH-rhodamine snython will be attached. In some embodiments, the linkinggroup is attached at the C2′, C2″, C4′, C5′, C7′, C7″, C5, or C6position of the N-protected NH-rhodamine synthon. In a specificembodiment, the linking group is attached at the C4′, C5′, C5 or C6position.

The N-protected NH-rhodamine snython can include a single linking groupLG, or it can include more than one linking group LG. In embodimentsthat employ more than one linking group, the linking groups may be thesame, or they may be different. N-protected NH-rhodamine synthons thatinclude multiple linking groups LG that are different from one anothercan have different groups or moieties attached to different positions ofthe parent NH-rhodamine ring using orthogonal chemistries.

The identity of a linking group may, in some instances, depend upon itslocation on the parent NH-rhodamine ring. In some embodiments in whichthe linking group LG is attached at the C4′-or C5′-position of theparent NH-rhodamine ring, the linking group LG is a group of the formula—(CH₂)_(n)—F^(y), where n is an integer ranging from 0 to 10 and F^(y)is as described above. In a specific embodiment, n is 1 and F^(y) is—NH₂.

In some embodiments in which the linking group LG is attached at the 5-or 6-position of the parent NH-rhodamine ring, the linking group LG is agroup of the formula —(CH₂)_(n)—C(O)OR^(f), where R^(f) is selected fromhydrogen and a good leaving group and n is as previously defined. Insome specific embodiments, the linking group LG comprises an NHS ester.In some specific embodiments, n is 0 and R^(f) is NHS.

In some embodiments, the N-protected NH-rhodamine comprising the labelmoiety of the various reagents described herein is described bystructural formulae (IIIc), (IIIb) or (IIIc); below:

wherein:

-   -   R′ is selected from R^(3′) and hydrogen;    -   R″ is selected from R^(6′) and hydrogen;    -   R⁹ is an acyl protecting group, optionally of the formula        —C(O)R¹⁰, where R¹⁰ is as previously defined;    -   R^(1′), R^(2′), R^(2″), R^(4′), R^(4″), R^(5′), R^(5″), R^(7′),        R^(7″) and R^(8′), when taken alone, are each, independently of        one another, selected from hydrogen lower alkyl, (C6-C14) aryl,        (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered        heteroarylalkyl, —R^(b) and —(CH₂)_(x)—R^(b), where x and R^(b)        are as previously defined, or, alternatively, R^(1′) and R^(2′)        or R^(7′) and R^(8′) are taken together with the carbon atoms to        which they are bonded to form a benzo group and/or R^(4′) and        R^(4″) and/or R^(5′) and R^(5″) are taken together with the        carbon atoms to which they are bonded to form a benzo group;

R^(3′) and R^(6′), when taken alone, are each, independently of oneanother, selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl,5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, oralternatively, R^(3′) and R^(2′) or R^(4′) and/or R^(6′) and R^(5′) orR^(7′) in the compounds of structural formula (IIIa), R^(3′) and R^(2′)or R^(4′) and/or R^(6′) and R^(5′) or R^(7″) in the compounds ofstructural formula (IIIb), or R^(3′) and R^(2″) or R^(4′) and/or R^(6′)and R^(5′) or R^(7″) in the compounds of structural formula (IIIc) aretaken together with the atoms to which they are bonded to form a 5- or6-membered saturated or unsaturated ring that may optionally includefrom 1 to 4 additional heteroatoms (typically selected from O, N and S)and that is optionally sutstituted with one or more of the same ordifferent lower alkyl, benzo or pyrido groups;

and R⁴, R⁵, R⁶, and R⁷ are each, independently of one another, selectedfrom hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 6-14membered heteroaryl, 7-20 membered heteroarylalkyl, —R^(b) and—(CH₂)_(x)—R^(b), where x and R^(b) are as previously defined,

with the proviso that at least one of R^(2′), R^(4′), R^(5′), R^(7′), R⁵or R⁶ in the compounds structural formula (IIIa), at least one ofR^(2′), R^(4′), R^(5′), R^(7″), R⁵ or R⁶ in the compounds of structuralformula (IIIb) and at least one of R^(2″), R^(4′), R^(5′), R^(7″), R⁵ orR⁶ in the compounds of structural formula (IIIc) comprises a group ofthe formula —Y—, where Y represents a portion of a linkage contributedby a linking group comprising a functional group F^(y), as describedabove.

In some embodiments, the N-protected NH-rhodamine comprising the labelmoiety of the various reagents described herein excludes 4,7-dichloroR6G (5- and/or 6-isomers) and/or rhodamines described by structuralformula (IIIa) in which:

-   -   R′ and R″ are each ethyl;    -   R^(1′), R^(4′), R^(5′) and R^(6′) are each hydrogen;    -   R^(2′) and R^(7′) are each methyl;    -   R⁴ and R⁷ are each chloro; and    -   one of R⁵ or R⁶ is hydrogen and the other is —C(O)—.

In some embodiments, the N-protected NH-rhodamine moieties according tostructural formulae (IIIa), (IIIb) and (IIIc), are, respectively,selected from moieties defined by structural formulae (IIIa.1),(IIIa.2), (IIIb.1), (IIIb.2), (IIIc.1) and (IIIc.2), below:

wherein R′, R″, R^(1′), R^(2′), R^(2″), R^(3′), R^(4′), R^(4′), R^(5′),R^(5″), R^(6′), R^(7′), R^(7″), R^(8′), R⁴, R⁵, R⁶, R⁷, R⁹ and Y are aspreviously defined for structural formulae (Ma), (IIIb) and (IIIc).

Specific exemplary embodiments of moieties defined by structuralformulae (IIIa), (IIIa.1), (IIIa.2), (IIIb), (IIIb.1), (IIIb.2), (IIIc),(IIIc.1) and (IIIc.2) include structures that have one or moreapplicable features selected from:

(i) Y is selected from —C(O)—, —S(O)₂—, —S— and —NH—;

(ii) R⁴ and R⁷ are each chloro;

(iii) R^(1′) and R^(8′) are each hydrogen;

(iv) R^(1′) and R^(2′) or R^(7′) and R^(8′) are taken together to form abenzo group;

(v) R^(2′) and R^(7′) are each hydrogen or lower alkyl;

(vi) R′ is R^(3′) and R″ is R^(6′);

(vii) R′ is R^(3′), R″ is R^(6′), and R^(3′) and R^(6′) are takentogether with a substituent group on an adjacent carbon atom to form agroup selected from —CH₂CH₂—, —CH₂CH₂CH₂—, —C(CH₃)₂CH═C(CH₃)—,—C(CH₃)₂CH═CH—, —CH₂—C(CH₃)₂— and

As discussed previously, the label moiety can comprise one or moreadditional dyes such that the N-protected NH-rhodamine, oncedeprotected, is a member of a larger, energy transfer dye network. Suchenergy transfer dye networks are well-known in the art, and includecombinations of fluorescent dyes whose spectral properties are matched,and/or whose relative distances to one another are adjusted, so that onefluorescent dye in the network, when excited by incident irradiation ofan appropriate wavelength, transfers its excitation energy to anotherfluorescent dyes in the network, which then transfers its excitationenergy to yet another fluorescent dye in the network, and so forth,resulting in fluorescence by the ultimate acceptor dye in the network.Dye networks provide label moieties having long Stoke's shifts. In suchnetworks, fluorophores that transfer, or donate, their excitation energyto another fluorphore in the network are referred to as “donors.”Fluorophores that receive, or accept, excitation energy from anotherfluorophore are referred to as “acceptors.” In dye networks containingonly two fluorescent dyes, one acts as the donor and the other as theacceptor. In dye networks containing three or more fluorescent dyes, atleast one dye acts as both a donor and acceptor. The principles of howdye networks work, as well as the criteria for selecting and linkingindividual dyes suitable for creating such networks are well known, andare described, for example, in Hung et al., 1997, Anal. Biochem.252:78-88.

In the label moieties described herein that comprise dye networks, theN-protected NH-rhodamine dye, once deprotected, may act as a donor or anacceptor, or as both a donor and acceptor, depending upon the identitiesof the other dyes comprising the network and the desired incident andfluorescent wavelengths. A vast number of dyes suitable for use asdonors and/or acceptors for NH-rhodamine dyes are known in the art, andinclude by way of example and not limitation, xanthene dyes (such as,for example, fluorescein, rhodamine and rhodol dyes), pyrene dyes,coumarin dyes (for example, hydroxy- and amino-coumarins), cyanine dyes,phthalocyanine dyes and lanthenide complexes. Specific, non-limitingexamples of these dyes in the context of energy transfer dye networksare described in Hung et al., 1996, Anal. Biochem. 238:165-170; Medintzet al., 2004, Proc. Nat'l Acad. Sci. USA 101(26):9612-9617; U.S. Pat.No. 5,800,996; Sudhaker et al., 2003, Nucleosides, Nucleotides & NucleicAcids 22:1443-1445; U.S. Pat. No. 6,358,684; Majumdar et al., 2005, J.Mol. Biol. 351:1123-1145; Dietrich et al., 2002, Reviews Mol.Biotechnology 82(3):211-231; Tsuji et al., 2001, Biophysical J.81(1):501-515; Dickson et al., 1995, J. Photochemistry & Photobiology27(1):3-19; and Kumar et al., 2004, Developments in Nucl. Acid Res.1:251-274, the disclosures of which are incorporated herein byreferences. Any of these dyes that can be suitably protected inaccordance with the principles desribed herein can be used as donor andacceptor dyes in label moieties that comprise dye networks. In someembodiments, one or more of the donor and/or acceptor dyes comprisingthe network can be an N-protected NH-rhodamine dye as described herein.Specific positions for attaching donor and/or acceptor dyes to rhodaminedyes to form dye networks, as well as specific linkages and linkersuseful for attaching such dyes, are well-known in the art. Specificexamples are described, for example, in U.S. Pat. Nos. 6,811,979;6,008,379; 5,945,526; 5,863,727; and 5,800,996, the disclosures of whichare incorporated herein by reference.

In some embodiments, the linker linking the donor and acceptor dyes isan anionic linker as described in U.S. Pat. No. 6,811,979, thedisclosure of which is incorporated herein by reference (see, e.g., thedisclosure at Col. 17, line 25 through Col. 18, line 37 and FIGS. 1-17).

In some embodiments of the reagents described herein, the label moietyincludes a donor dye for the NH-rhodamine dye. In some embodiments, thedonor dye is a fluorescein or rhodamine dye, such as, for example, oneof the NH-rhodamine dyes described herein. In a specific embodiment, thedonor dye is a fluorescein dye. Fluorescein dyes are similar instructure to rhodamine dyes, with the exception that the 3- and6-positions of the parent xanthene ring (corresponding to the 3′- and6′-positions of the NH-rhodamine rings of structural formulae (Ia), (Ib)and (Ic)), are substituted with a hydroxyl groups. Like the rhodamines,the fluoresceins can also have extended ring structures in which thecarbon atoms at positions C3 and C4 and/or C5 and C6 of the parentxanthene ring are included in aryl bridges such as benzo groups. Thus,the fluoresceins generally include compounds according to structuralformulae (IVa), (IVb) and (IVc), below:

Like the NH-rhodamines, the carbons at positions C1′, C2′, C2″, C4′,C4″, C5′, C5″, C7′, C7″, C8′, C4, C5, C6 and C7 of the fluoroesceinrings of structural formulae (IVa), (IVb) and (IVc) can be substitutedwith a variety of different substituents, such as those describedpreviously for the NH-rhodamines.

When included in the label moieties described herein, the hydroxyls atthe C3′ and C6′ positions should be protected with protecting groupshaving the same general properties as the groups protecting theexocyclic amines of the NH-rhodamines, discussed above. Thus, inspecific embodiments the protecting groups are stable to the conditionsused to synthesize oligonucleotides, such as the conditions used tosynthesize and oxidize oligonucleotides via the phosphite triestermethod, and are labile under the conditions typically used to deprotectand/or cleave synthetic oligonucleotides from the synthesis resin, suchas, for example, incubation in concentrated ammonium hydroxide at roomtemperature or 55° C.

A wide variety of protecting groups having suitable properties are knownin the art, and include by way of example and not limitation, the acylgroups described above in connection with N-protected NH-rhodamine dyes.In a specific embodiment, the protecting group is of the formula—C(O)—R¹⁰, where R¹⁰ is as previously defined. In some embodiments, R¹⁰is t-butyl. Fluoresceins in which the C3′ and C6′ exocyclic hydroxylsinclude protecting groups are referred to herein as “O-protectedfluoresceins.” O-protected fluoresceins corresponding to thefluoresceins of structural formulae (IVa), (IVb) and (IVc),respectively, are illustrated as structural formulae (Va), (Vb) and(Vc), below:

wherein R⁹ represents the protecting group.

A vast variety of different fluorescein dyes that can be suitablyprotected and incorporated into label moieties for use as a donors forthe NH-rhodamine moiety are known in the art. Specific exemplaryfluorescein dyes are described, for example, in U.S. Pat. Nos.6,221,604; 6,008,379; 5,840,999; 5,750,409; 5,654,441; 5,188,934;5,066,580; 4,481,136; 4,439,356; WO 99/16832; and EP 0 050 684, thedisclosures of which are incorporated herein by reference. Skilledartisans will be able to select a fluorescein having spectral propertiessuitable for use as a donor for a specific NH-rhodamine. Specificembodiments of parent fluoroescein dyes that may be incorprated in thelabel moieties of the reagents described herein are illustrated in FIG.1C.

The donor and N-protected NH-rhodamine acceptor can be linked to oneanother in a variety of orientations, either directly or with the aid ofa linker. In some embodiments in which the donor is an O-protectedfluorescein or an N-protected NH-rhodamine, the donor is linked to the2′-, 2″-, 4′-, 5′-, 7′-, 7″-, 5- or 6-position of the N-protectedNH-rhodamine acceptor via its 2′-, 2″-, 4′-, 5′-, 7′-, 7″-, 5- or6-position.

Specific exemplary linkage orientations are provided in Table 2, below:

TABLE 2 donor/acceptor acceptor/donor nickname 4’- or 5’- 4’- or 5’-head-to-head 4’- or 5’- 5- or 6- head-to-tail 5- or 6’ 5- or 6-tail-to-tail 2’-, 2”-, 7’- or 7”- 2’-, 2”-, 7’- or 7”- side-to-side 2’-,2”-, 7’- or 7”- 4’- or 5’- side-to-head 2’-, 2”-, 7’- or 7”- 5- or 6-side-to-tail

Label moieties comprising dye networks, such as the donor-acceptor dyenetworks of Table 2, can be linked to the remainder of the reagent atany available position. In some embodiments, label moieties comprisinghead-to-head linked acceptor/donor pairs are attached to the remainderof the reagent via the 5- or 6-position of the donor or acceptor moiety.In some embodiments, label moieties comprising head-to-tail linkedacceptor/donor pairs are attached to the remainder of the reagent via anavailable 4′-, 5′-, 5- or 6-position of the donor or acceptor moiety. Insome embodiments, label moieties comprising tail-to-tail linkedacceptor/donor pairs are attached to the remainder of the reagent viathe 4′- or 5′-position of the donor or acceptor. In some embodiments,label moieties comprising side-to-side linked acceptor/donor pairs areattached to the remainder of the reagent via the 4′-, 5′-, 5- or6-position of the donor or acceptor. In some embodiments, label moietiescomprising side-to-head linked acceptor/donor pairs are attached to theremainder of the reagent via an available 4′-, 5′-, 5- or 6-position ofthe donor or acceptor. In some embodiments, label moieties comprisingside-to-tail linked acceptor/donor paris are attached to the remainderof the reagent via an available 4′-, 5′-, 5- or 6-position of the donoror acceptor.

Regardless of their orientation, the O-protected fluorescein orN-protected NH-rhodamine donor and the N-protected NH-rhodamine acceptorare typically linked to one another via a linker. It has been discoveredpreviously that it may be advantageous to link such donor and acceptordyes via linkers that are rigid in nature and/or that are relativelylong, for example, in the range of approximately 12-20 Å in length (asused herein, the “length” of a linker refers to the distance between thelinked moieties as determined by calculating the sum of the lengths ofthe chemical bonds defining the shortest continuous path between themoieties). Without intending to be bound by any theory of operation, itis believed that linkers that tend to hold the donor and acceptor inclose proximity to one another without permitting their chromophores totouch one another yield suitably efficient energy transfer. In thisregard, the rigidity and length of the linker are coupled parameters.Generally, shorter linkers (for example linkers having a length of about5 to 12 Å) should include a greater degree of rigidity. Longer linkers(for example linkers having a length in the range of about 15 to 30 Å)can include a lesser degree of rigidity, or even no rigidity. Short,non-rigid (floppy) linkers should be avoided.

Rigidity can be achieved through the use of groups that have restrictedangles of rotation about their bonds, for example, through the use ofarylene or heteroarylene moieties, and/or alkylene moieties thatcomprise double and/or triple bonds. A variety of linkers useful forlinking rhodamine and fluorescein dyes to one another in the context ofenergy transfer dyes are known in the art, and are described, forexample, in U.S. Pat. No. 5,800,996, the disclosure of which isincorporated herein by reference. Specific examples of linkers usefulfor linking 0-protected fluorescein or N-protected NH-rhodamine donorsto N-protected NH-rhodamine acceptors in the label moieties describedherein include, by way of example and not limitation, groups of theformula:

—Z—(CH₂)_(a)—[(Ar)_(b)—(CH₂)_(a)]_(c)—Z—;   (L.1)

—Z—(CH₂)_(a)—[C≡C—(CH₂)_(a)]_(c)—Z—;   (L.2)

—Z—(CH₂)_(a)—[C≡C—(Ar)_(b)]_(c)—(CH₂)_(a)—Z—;   (L.3)

—Z—(CH₂)_(d)—NH—C(O)—[(CH₂)_(a)—(Ar)—(CH₂)_(a)—C(O)—NH]_(c)—(CH₂)_(d)—Z—;and   (L.4)

—Z—[CH₂(CH₂)_(e)O]_(f)—CH₂(CH₂)_(e)O—,   (L.5)

where each Z represents, independently of the others, a portion of alinkage contributed by a linking group F^(z), as previously described,each a represents, independently of the others, an integer ranging from0 to 4; each b represents, independently of the others, an integerranging from 1 to 2; each c represents, independently of the others, aninteger ranging from 1 to 5; each d represents, independently of theothers, an integer ranging from 1 to 10; each e represents,independently of the others, an integer ranging from 1 to 4; each frepresents, independently of the others, an integer ranging from 1 to10; and each Ar represents, independently of the others, an optionallysubstituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene,arylene or heteroarylene group. Non-limiting exemplary embodiments of Arinclude groups derived from lower cycloalkanes, lowercycloheteroalkanes, parent aromatic ring systems and parentheteroaromatic ring systems, as described previously. Specific,non-limiting exemplary embodiments of Ar include cyclohexane,piperazine, benzene, napthalene, phenol, furan, pyridine, piperidine,imidazole, pyrrolidine and oxadizole. Specific, non-limiting exemplaryembodiments of linkers are illustrated in FIG. 2 . In FIG. 2 , Z¹ and Z²each represent, independently of one another, a portion of a linkagecontributed by a functional group F^(z), as previously described, and Kis selected from —CH— and —N—. In some specific embodiments of thelinkers illustrated in FIG. 2 , one of Z¹ or Z² is —NH— and the other isselected from —O—, —C(O)— and —S(O)₂—.

In some embodiments, the linker linking the donor and acceptor dyes isan anionic linker as described in U.S. Pat. No. 6,811,979, thedisclosure of which is incorporated herein by reference (see, e.g., thedisclosure at Col. 17, line 25 through Col. 18, line 37 and FIGS. 1-17). Specific, non-limiting exemplary embodiments of suitable anioniclinkers include the linkers of formulae (L.1) through (L.4), above, inwhich one or more of the Ar groups are substituted with one or moresubstitent groups having a negative charge under the conditions of use,such as, for example, at a pH in the range of about pH 7 to about pH 9.Specific, non-limiting examples of suitable substituent groups includephosphate esters, sulfate esters, sulfonate and carboxylate groups.

In some embodiements, the label moiety is of the formula (VI):

A-Z¹-Sp-Z²-D   (VI)

where A represents the N-protected NH-rhodamine acceptor, D representsthe donor, for example, an N-protected NH-rhodamine or O-protectedfluorescein donor, Z¹ and Z² represent portions of linkages provided bylinking moieties comprising a functional group F^(z), as previouslydescribed, and Sp represents a spacing moiety, as previously described.In some specific embodiments, A is selected from structural formulaeA.1, A.2, A.3, A.4, A.5 and A.6 and D is selected from structuralformulae D.1, D.2, D.3, D.4, D.5 and D.6, illustrated below. In somespecific embodiments, A is selected from structural formulae A.7, A.8,A.9, A.10, A.11 and A.12 and D is selected from structural formulae D.7,D.8, D.9, D.10, D.11 and D.12, illustrated below.

In structural formulae A.1-A.12 and D.1-D.12:

E¹ is selected from —NHR⁹, —NR^(3′)R⁹ and —OR^(9b);

E² is selected from —NHR⁹, —NR^(6′)R⁹ and —OR^(9b);

R^(9b) is R⁹;

Y^(1a), Y^(1b), Y^(2a), Y^(2b), Y^(3a) and Y^(3b) are each,independently of one another, selected from —O—, —S—, —NH—, —C(O—) and—S(O)₂—; and

R′, R″, R^(1′), R^(2′), R^(2″), R^(3′), R^(4′), R^(4″), R^(5′), R^(5″),R^(6′), R^(7′), R^(7″), R^(8′), R⁴, R⁵, R⁶ and R⁷ are as previouslydefined for structural formulae (IIIa), (IIIb) and (IIIc), with theproviso that when E¹ and E² are —OR^(9b), then R^(1′) and R^(2′) andR^(7′) and R^(8′) may both include benzo and/or pyrido groupssimultanously.

In some specific embodiments of label moieties according to structuralformula (VI), Y^(1a), Y^(2a) and Y^(3a) are —NH—; Y^(1b), Y^(2b) andY^(3b) are selected from —C(O)— and —S(O)₂—; Z¹ is selected from —C(O)—and —S(O)₂—; Z² is —NH— and Sp is a group selected from:

—(CH₂)_(a)—[(Ar)_(b)—(CH₂)_(a)]_(c)—;   (Sp.1)

—(CH₂)_(a)—[C≡C—(CH₂)_(a)]_(c)—;   (Sp.2)

—(CH₂)_(a)—[C≡C—(Ar)_(b)]_(c)—(CH₂)_(a)—;   (Sp.3)

—(CH₂)_(d)—NH—C(O)—[(CH₂)_(a)—(Ar)—(CH₂)_(a)—C(O)—NH]_(c)—(CH₂)_(d)—;and

—[CH₂(CH₂)_(e)O]_(f)—CH₂(CH₂)—,   (Sp.5)

where a, b, c, d, e,fand Ar are as previously defined.

In some specific embodiments of label moieties according to structuralformula (VI), R⁹ is selected from —C(O)CH₃ and C(O)CF₃ and R^(9a) is—C(O)C(CH₃)₃.

5.5 Phosphate Ester Precursor Group

Many embodiments of the reagents described herein include a phosphateester precursor group (“PEP”). When used in a step-wise synthesis tosynthesize a labeled oligonucleotide, the PEP group is coupled to anyavailable hydroxyl group, which may be the 5′-hydroxyl group of anascent synthetic oligonucleotide, ultimately contributing, after anyrequired oxidation and/or deprotection steps, a linkage linking thelabel moiety to the synthetic oligonucleotide. The linkage formed may bea phosphate ester linkage or a modified phosphate ester linkage as isknow in the art.

A variety of different groups suitable for coupling reagents to primaryhydroxyl groups to yield phosphate ester or modified phosphate esterlinkages are well-known in the art. Specific examples include, by way ofexample and not limitation, phosphoramidite groups (see, e.g., Letsingeret al., 1969, J. Am. Chem. Soc. 91:3350-3355; Letsinger et al., 1975 J.Am. Chem. Soc. 97:3278; Matteucci & Caruthers, 1981, J. Am. Chem. Soc.103:3185; Beaucage & Caruthers, 1981, Tetrahedron Lett. 22:1859; thedisclosures of which are incorporated herein by reference),2-chlorophenyl- or 2,5-dichlorophenyl-phosphate groups (see, e.g.,Sproat & Gait, “Solid Phase Synthesis of Oligonucleotides by thePhosphotriester Method,” In: Oligonucleotide Synthesis, A PracticalApproach, Gait, Ed., 1984, IRL Press, pages 83-115), the disclosures ofwhich are incorporated herein by reference), and H-phosphonate groups(see, e.g., Garegg et al., 1985, Chem. Scr. 25:280-282; Garegg et al.,1986, Tet. Lett. 27:4051-4054; Garegg et al. 1986, Tet. Lett.27:4055-4058; Garegg et al., 1986, Chem. Scr. 26:59-62; Froehler &Matteucci, 1986, Tet. Lett. 27:469-472; Froehler et al., 1986, Nucl.Acid Res. 14:5399-5407, the disclosures of which are incorporated hereinby reference). In a specific embodiment, the PEP group is aphosphoramidite group of the formula (P.1):

wherein:

-   -   R²⁰ is selected from a linear, branched or cyclic saturated or        unsaturated alkyl containing from 1 to 10 carbon atoms,        2-cyanoethyl, an aryl containing from 6 to 10 ring carbon atoms        and an arylalkyl containing from 6 to 10 ring carbon atoms and        from 1 to 10 alkylene carbon atoms; and

R²¹ and R²² are each, independently of one another, selected from alinear, branched or cyclic, saturated or unsaturated alkyl containingfrom 1 to 10 carbon atoms, an aryl containing from 6 to 10 ring carbonatoms and an arylalkyl containing from 6 to 10 ring carbon atoms andfrom 1 to 10 aklylene carbon atoms, or, alternatively, R²¹ and R²² aretaken together with the nitrogen atom to which they are bonded to form asaturated or unsaturated ring that contains from 5 to 6 ring atoms, oneor two of which, in addition to the illustrated nitrogen atom, can beheteroatom selected from O, N and S.

In a specific embodiment, R²⁰ is 2-cyanoethyl and R²¹ and R²² are eachisopropyl.

5.6 Synthesis Handles

Many embodiments of the reagents described herein include one or moresynthesis handles that provide, after suitable deprotection, ifnecessary, sites that can be used for the attachment of additionalgroups or moieties to the synthetic labeled oligonucleotide. The groupscan be attached to a synthesis handle during the course of synthesizingthe labeled oligonucleotide, or, alternatively, the synthesis handle canbe deprotected post-synthesis to reveal a functional group to whichadditional groups or moieties can be attached. For example, a synthesishandle could comprise a primary amine group that is protected with aprotecting group that is stable to the conditions used to carry out thesynthesis of the labeled oligonucleotide. Removal of the protectinggroup following synthesis, either concurrently with, or separately from,the removal of the various other protecting groups on the syntheticoligonucleotide, provides a primary amino group to which additionalgroups and/or moieties can be attached.

A variety of different types of reactive groups protected withprotecting groups suitable for use in oligonucleotide synthesis areknown in the art, and include by way of example and not limitation,amino groups (protected with, for example, trifluroacetyl or4-monomethoxytrityl groups), hydroxyl groups (protected with, forexample, 4,4′-dimethoxytrityl groups), thiol groups (protected with, forexample, trityl or alkylthiol groups) and aldehyde groups (protectedwith, for example, an acetal protecting group). All of these protectedreactive groups can comprise the synthesis handle of the reagentsdescribed herein.

In some embodiments, the synthesis handle comprises a protected primaryhydroxyl of the formula —OR^(e), where R^(e) represents an acid-labileprotecting group that can be selectively removed during the course ofsynthesizing an oligonucleotide. Acid labile protecting groups suitablefor protecting primary hydroxyl groups in the context of oligonucleotidesynthesis are known in the art, and include, by way of example and notlimitation, triphenylmethyl (trityl), 4-monomethoxytrityl,4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, bis(p-anisyl)phenylmethyl, naphthyldiphenylmethyl,p-(p′-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl and 9-(9-phenyl-10-oxo)anthryl. All of thesegroups can be removed by treatment with mild acid, such as by treatmentwith 2.5% or 3% di- or trichloro acid and in dichoromethane. Methods ofprotecting primary hydroxyl groups with the above-listed acid-labileprotecting groups are well-known.

5.7 Solid Supports

Many embodiments of the reagents described herein comprise solidsupports to which the other moieties and/or groups are attached. Thesolid supports are typically activated with functional groups, such asamino or hydroxyl groups, to which linkers bearing linking groupssuitable for attachment of the other moieties are attached.

A variety of materials that can be activated with functional groupssuitable for attachment to a variety of moieties and linkers, as well asmethods of activating the materials to include the functional groups,are known in the art, and include by way of example, controlled poreglass, polystyrene and graft co-polymers. Any of these materials be usedas solid supports in the reagents described herein.

5.8 Synthesis Regents Useful for Terminal Hydroxyl Labeling

Some embodiments of the synthesis reagents described herein aredescribed by stuctural formula (VII):

LM-L-PEP   (VII)

where LM represents a label moiety as described herein, L represents anoptional linker as described herein and PEP represents a phosphate esterprecursor group as described herein. The reagents can include additionalgroups or moieties, such as synthesis handles. In some embodiments, thesynthesis reagents comprise a label moiety and a PEP group, and do notinclude additional moieties or groups. Such synthesis reagents can becoupled to a hydroxyl group during the step-wise synthesis of anoligonucleotide, and are useful for, among other things, attaching alabel moiety to a terminal hydroxyl group of a syntheticoligonucleotide, which is commonly the 5′-hydroxyl.

The PEP group can be attached directly to the label moiety, or it may beattached to the label moiety with the aid of a linker. As PEP groups aregenerally linked to molecules by coupling suitable reagents to primaryhydroxyl groups, in embodiments in which the PEP group is attacheddirectly to the label moiety, the label moitey should include asubstituent group that comprises a primary hydroxyl group. Inembodiments in which the PEP group is linked to the label moiety withthe aid of a linker, the linker synthon should include a linking groupsuitable for forming a linkage with a linking group on the label moietysynthon and a primary hydroxyl group suitable for attachment to the PEPgroup. Suitable linker synthons include, but are not limited to,synthons of the formula F-Sp-OH, where FZ is a functional groupcomplementary to a functional group on the label moiety synthon and Sprepresents a spacing moiety. The spacing moiety can comprise anycombination of atoms and/or functional groups stable to the conditionsthat will be used to synthesize and deprotect the labeled syntheticoligonucleotide. Non-limiting exemplary linkers are illustrated in FIG.2 , where Z² is O. In some embodiments, Sp is an optionally substitutedalkylene chain that contains from 1 to 10 chain atoms. In a specificembodiment, Sp is an unsubstitued alkylene chain containing from 1 to 9carbon chain atoms.

In some embodiments, the synthesis reagents are compounds according tostructural forumula (VII) in which:

-   -   LM is one of the embodiments of label moieties specifically        exemplified above;    -   L is selected from —Z—(CH₂)₃₋₆—O—,        —Z—(CH₂)_(a)—[(Ar)_(b)—(CH₂)_(a)]_(c)—O—,        —Z—(CH₂)_(a)—[C≡C—(CH₂)_(a)]_(c)—O—,        —Z—(CH₂)_(a)—[C≡C—(Ar)_(b)]_(c)—(CH₂)_(a)—O—,        —Z—(CH₂)_(d)—NH—C(O)—[(CH₂)_(a)—(Ar)—(CH₂)_(a)—C(O)—NH]_(c)—(CH₂)_(d)—O—,        —Z—[CH₂(CH₂)_(e)O]_(f)—CH₂(CH₂)_(e)O— and one of the linkers        illustrated in FIG. 2 in which Z² is O; and

PEP is a phosphoramidite group, such as for example, a phosphoramiditegroup of structural formula P.1, as described above. In some specificembodiments, Z in linker L is —NH—.

In some embodiments, the linker in synthesis reagents according tostructural formula (VII) comprises a nucleoside, such that the synthesisreagent is nucleosidic. In some embodiments, nucleosidic synthesisreagents are compounds according to structural formula (VII.1):

where PEP represents the phosphate ester precursor group, B represents anucleobase, LM represents the label moiety and L² represents a linkerlinking nucleobase B to linker LM. The features and properties ofnucleobase B and linker L are decribed in more detail, below.Non-limiting exemplary nucleosidic synthesis reagents according tostructural formula (VII.1) are illustrated in FIG. 4 .

An exemplary scheme for synthesizing embodiments of synthesis reagentsin which the PEP group is linked to the label moiety via an optionallinker is provided in Scheme (I), below, where the various R, F^(y),F^(z), Y, Z and S_(p) groups are as previously defined:

In Scheme (I) parent NH-rhodamine synthon 100, which includes a linkinggroup that comprises functional group F^(y), is acetylated withanhydride 101 to yield N-acetyl-protected NH-rhodamine synthon 102.Synthon 102 is then coupled to linker synthon 103 to yield compound 104.Depending upon the identity of F^(y), synthon 102 may require activationprior to coupling. For example, if F^(y) is a carboxyl group, it can beactivated as an ester, such as an NHS ester, prior to coupling. Incompound 104, —Y—Z— represents the linkage formed by complementaryfunctional groups F^(y) and F^(z), where Y represents the portioncontributed by F^(y) and Z represents the portion contributed by F^(z),as previously described. Compound 104 is then reacted with PEP synthon105, which in the specific embodiment illustrated is a phosphine, toyield phosphoramidite synthesis reagent 106.

5.9 Synthesis Reagents Useful for Internal or 3′-End Labeling

The synthesis reagents described herein may optionally include one ormore synthesis handles useful for the attachment of additional groupsand/or moieties. Synthesis reagents that include a synthesis handle ofthe formula —OR^(e), where R^(e) represents an acid-labile protectinggroup as previously described, provide a primary hydroxyl group to whichadditional nucleotides can be attached. As a consequence, synthesisreagents that include such a synthesis handle can be used to labelsynthetic oligonucleotides at the 5′-hydroxyl, the 3′-hydroxyl or at oneor more internal positions. They can also be coupled to one another, orto other phosphoramidite labeling reagents, permitting the synthesis ofoligonucleotides containing a plurality of label moieties.

The label moiety, PEP group and synthesis handle —OR^(e) comprising thesynthesis reagent can be linked together in any fashion and/ororientation that permits them to perform their respective functions. Asa specific example, the PEP group and synthesis handle can each belinked to the label moiety, optionally via linkers. In some embodiments,such synthesis reagents are compounds according to structural formula(VIII):

R^(e)O-L-LM-L-PEP   (VIII)

where each L represents, independently of the other, an optional linker,LM represents the label moiety and PEP represents the phosphate esterprecursor group. Non-limiting examples of suitable protecting groupsR^(e), linkers L, label moieties LM and phosphate ester precursor groupsinclude those specifically exemplifed above.

As another specific example, the PEP group and synthesis handle —OR^(e)may be attached to a branched linker that is attached to the labelmoiety. In some embodiments, such synthesis reagents are compoundsaccording to structural formula (IX):

where L represent a linker, LM represents the label moiety and PEPrepresents the phosphate ester precursor group.

In a specific embodiment, synthesis reagents according to structuralformula (IX) are compounds according to structural formula (IX.1):

where LM represents the label moiety, —Z— represents a portion of alinkage contributed by a functional F^(z) on the linker, Sp¹, Sp² andSp³, which can be the same or different, each represent spacingmoieties, G represents CH, N, or a group comprising and arylene,phenylene, heteroarylene, lower cycloalkylene, cyclohexylene, and/orlower cycloheteroalkylene, and PEP represents the phosphate esterprecursor group. In some embodiments, LM is one of the embodiments oflabel moities specifically exemplified above, Sp¹, Sp² and Sp³ are each,independently of one another, selected from an alkylene chain containingfrom 1 to 9 carbon atoms, Sp.1, Sp.2, Sp.3, Sp.4 and Sp.5 (definedabove), and/or PEP is a phosphoramidite group according to structuralformula P.1, supra. Non-limiting specific embodiments of exemplarysynthesis reagents according to structural formula (IX.1) areillustrated in FIGS. 3 and 4 .

In some embodiments, the synthesis handle —OR^(e) is provided by anucleoside, such that the synthesis reagent is nucleosidic. In suchnucleosidic synthesis reagents, the label moiety is typically linked tothe nucleobase of the nucleoside by way of a linker, and any exocyclicfunctional groups on the nucleobase that are reactive under theconditions used to synthesize the labeled oligonucleotide, such as, forexample, exocyclic amines, are protected.

The nucleoside can be any nucleoside that can be suitably protected foruse in the synthesis of oligonucleotides, and may comprise a2′-deoxyribose sugar moiety, a 3′-deoxyribose sugar moiety (useful forsynthesizing labeled oligonucleotides including a 2′-5′ internucleotidelinkage), a suitably protected ribose moiety, a substituted version ofany of these ribose moieties, or even a non-ribose sugar moiety.

In some embodiments, such nucleosidic synthesis reagents are compoundsaccording to structural formulae (IX.2), (IX.3), (IX.4) and (IX.5):

wherein LM represents the label moiety, B represents a suitablyprotected nucleobase, L² represents a linker linking the label moiety tothe nucleobase, R^(e) represents the acid-labile protecting group, PEPrepresents the phosphate ester precursor group, O is an oxygen atom and,in structural formula (IX.4), R¹¹ represents a 2′-hydroxyl protectinggroup.

In the synthesis reagents according to structural formulae (VII.1),(IX.2), (IX.3), (IX.4) and (IX.5), the nucleobase B can be virtually anyheterocycle useful for incorporation into oligonucleotides. For example,the nucleobase may be one of the genetically encoding purines (adenineor guanine), one of the genetically encoding pyrimidines (cytosine,uracil or thymine), anologs and/or derivatives of the geneticallyencoding purines and/or pyrimidines (e.g., 7-deazadenine,7-deazaguanine, 5-methylcytosine), non-genetically encoding purinesand/or pyrimidines (e.g., inosine, xanthene and hypoxanthene) or othertypes of heterocycles. A wide variety of heterocycles useful forincorporating into oligonucleotides are known in the art and aredescribed, for example, in Practical Handbook of Biochemistry andMolecular Biology, Fasman, Ed., 1989, CRC Press (see, e.g., pages385-393 and the references cited therein), the disclosures of which areincorporated herein by reference. All of these various heterocycles, aswell as those that are later discovered, can be included in thenucleosidic synthesis reagents described herein.

When B is a purine in the synthesis reagents according to structuralformulae (VII.1), (IX.2), (IX.3), (IX.4) and (IX.5), the illustratedsugar moiety is typically attached to the N9 position of the purine, andwhen B is a pyrimidine, the illustrated sugar moiety is typicallyattached at the N1 position of the pyrimidine. Attachment sites forother nucleobases will be apparent to those of skill in the art.

Any exocyclic amine or other reactive group(s) on the nucleobase areprotected with protecting groups that are stable to the synthesisconditions used to synthesize the labeled oligonucleotide. A variety ofgroups that are suitable for protecting the exocyclic amine groups ofnucleoside nucleobases in the context of oligonucleotide synthesis arewell-known in the art, as are methods of preparing such protectednucleosides.

For example, groups that have been used to protect the exocyclic amineof adenine include benzyol (Bz), phenoxyacetyl (Pac) and isobutyryl(iBu). Groups that have been used to protect the exocyclic amine ofcystosine include acetyl (Ac) and Bz. Groups that have been used toprotect the exocyclic amine of guanine include iBu, dimethylformamide(Dmf) and 4-isopropyl-phenoxyacetyl (iPr-Pac). All of these protectinggroups can be removed by treatment with ammonium hydroxide at 55-65° C.for 2-3 hr. However, certain of these protecting groups can be removedunder milder conditions. For example, cleavage of the protecting groupsfrom A^(iBu), A^(Pac), C^(Ac) and G^(iPr-Pac) can be effected in 4-17hrs at room temperature with ammonium hydroxide, or with 0.05M potassiumcarbonate in methanol, or treatment with 25% t-butylamine in H₂O/EtOH.As some of the NH-rhodamine and/or other dyes comprising the reagentsdescribed herein may not be stable to the harsher deprotectionconditions required by other protecting groups, nucleosidic reagentswhich utilize protecting groups that can be removed under these milderdeprotection conditions are preferred.

The linker L² linking the label moiety LM to the nucleobase B may beattached to any position of the nucleobase. In some embodiments, when Bis a purine, the linker is attached to the 8-position of the purine,when B is a 7-deazapurine, the linker is attached to the 7-position ofthe 7-deazapurine, and when B is a pyrimidine, the linker is attached tothe 5-position of the pyrimidine.

In some embodiments, linkers L² useful for attaching LM to a nucleobasecomprise an acetylenic or alkenic amino linkage, such as, for example, alinkage selected from —C≡C—CH₂—NH—, —C≡C—C(O)—, —CH═CH—NH—,—CH═CH—C(O)—, —C≡C—CH₂—NH—C(O)—(CH₂)₁₋₆—NH—, and—CH═CH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, a propargyl-1-ethoxyamino linkage,such as, for example, a linkage having the formula—C≡CH—CH₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH— or a rigid linkage, such as forexample, a linkage selected from—C≡C—C≡C—CH₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH—,—C≡C—(Ar)₁₋₂—C≡C—CH₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH—,—C═C—(Ar)₁₋₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH— and—C≡C—(Ar)₁₋₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH—, where Ar is as definedpreviously.

In some embodiments, linkers L² useful for attaching LM to a purinenucleobase comprise an alkylamine, such as, for example, a linkage ofthe formula —NH—(CH₂)₁₋₆—NH—.

In some embodiments, linkers L² useful for attaching LM to a purine orpyrimidine nucleobse are anionic linkers as described in U.S. Pat. No.6,811,979, the disclosure of which is incorporated herein by reference(see, e.g., the disclosure at Col. 17, line 25 through Col. 18, line 37and FIGS. 1-17 ).

Methods of synthesizing nucleosides derivatized with linkers such asthose described above that are suitable for incorporating into thereagents described herein are described, for example, in Hobbs et al.,1989, J. Org. Chem. 54:3420; U.S. Pat. No. 5,151,507 to Hobbs et al.,U.S. Pat. No. 5,948,648 to Khan et al.; and U.S. Pat. No. 5,821,356 toKhan et al, the disclosures of which are incorporated herein byreference. The derivatized nucleosides can be used as synthons tosynthesize nucleosidic synthesis reagents as will be described in moredetail, below.

Specific exemplary embodiments of linker-dervatized nucleobases that maycomprise the nucleosidic reagents described herein are illustratedbelow:

Nucleosidic synthesis reagents can be prepared from linker-derivatizednucleoside synthons as illustrated in Scheme (II), below:

In Scheme (II), linker-derivatized nucleoside synthon 110 is protectedat the 5′-hydroxyl with an acid-labile protecting group, which isillustrated in the Scheme with exemplary chloride reagent R^(e)Cl, whereR^(e) is as previously defined. Treatment with base to remove thetrifluoroacetyl protecting group yields synthon 112. Reaction of synthon112 with label moiety synthon 102 (see Scheme (I), supra, followed bytreatment with PEP synthon 105, which in this specific exampleillustrated is a phosphine (see Scheme (I), supra) yields nucleosidicsynthesis reagent 114. Specific conditions for carrying out the varioussynthetic steps illustrated above are well known. Non-nucleosidicsynthesis reagents that include a synthesis handle, such as a synthesishandle of the formula —OR^(e), can be prepared by routine adaptation ofScheme (II).

5.10 Solid Support Reagents

Many embodiments of the reagents described herein include solidsupports. Such reagents generally comprise a solid support, a labelmoiety as described herein and a synthesis handle, and may includeadditional groups or moieties, such as additional label moieties,quenching moieties, synthesis handles and/or groups useful for, amongother things, stabilizing oligonucleotide duplexes, such as, forexample, agents that intercalate between base paris (intercalatingagents) and agent that bind the duplex minor groove (minor groovebinding, or MGB, agents). The solid support, label moiety, synthesishandle and any optional additional moieties may be linked to one anotherin any fashion or orientation that permits them to perform theirrespective functions.

In some embodiments, the solid support is attached to the remainder ofthe reagent via a linker. Linkers attaching solid supports to theremainder of the reagent typically include linkages that are selectivelycleavable under specified conditions such that, following synthesis, thesynthesized labeled oligonucleotide can be released from the solidsupport. In some embodiments, the linkages are labile to the conditionsused to deprotect the synthetic labeled oligonucleotide, such that theoligonucleotide is deprotected and cleaved from the solid support in asingle step. Such linkers typically include ester linkages, but mayinclude other linkages, such as, for example, carbonate esters,diisopropylsiloxy ethers, modified phosphates esters, etc.

Myriad selectively cleavable linkers useful in the context ofoligonucleotide synthesis are known in the art, as are methods ofderivatizing solid supports with such linkers. All of these variouslinkers can be adapted for use in the solid support reagents describedherein. Non limiting examples of solid support reagents comprisingexemplary linkers that are cleavable under the basic conditions used todeprotect synthetic oligonucleotides are are illustrated in FIG. 7 .

Like the synthesis reagents, the solid support reagents can benon-nucleosidic or nucleosidic in nature. Exemplary embodiments ofnon-nucleosidic solid support reagents include reagents according tostructural formula (X):

where LM represents the label moiety, L represents an optionalselectively cleavable linker and —OR^(e) represents the synthesishandle, where R^(e) is an acid-labile protecting group, as previouslydescribed.

In some embodiments, the solid support synthesis reagents of structuralformula (X) are non-nucleosidic reagents according to structural formula(X.1):

where Z, LM, G, Sp¹, Sp² and R^(e) are as previusly defined inconnection with structural formula (IX.1) and Sp⁴ represents aselectively cleavable spacing moiety. In some specific embodiments,selecteively cleavable spacing moiety Sp⁴ comprises an ester linkage.

In some embodiments, the solid support synthesis reagents of structuralformula (X) are nucleosidic reagents according to structural formuale(X.2), (X.3), (X.4) or (X.5):

wherein LM, R^(e), B. L² are as previously defined for structuralformulae (X.2), (X.3), (X.4) and/or (X.5), R¹¹ is as previously definedfor structural formula (IX.4) and Sp⁴ represents a selectively cleavablespacing moiety, as described above, which in some embodiments comprisesan ester linkage.

5.11 Additional Exemplary Embodiments

It is to be understood that the specific embodiments of the variousmoieties, groups and linkers described throughout the disclosure can beincluded in all of the reagents described herein. Moreover, the variousspecific embodiments can be combined with one another in any combinationas though the specific combination had been specifically exemplified. Asa specific example, any one of the specific embodiments of label moietyLM described herein can be included in any of the specificallyexemplified embodiemtns of non-nucleosidic and nucleosidic solid supportand synthesis reagents described herein. As another specific example,any one of the specific embodiments of phosphate ester precursor groupPEP, such as the phosphoramidite group of structural formula (P.1),supra, can be included in any of the synthesis reagents describedherein.

5.12 Uses of the Reagents

The various reagents described herein can be used in the step-wisesynthesis of oligonucleotides to synthesize oligonucleotides labeledwith rhodamine dyes directly on the synthesis resin. Thus, the variousreagents make available the ability to synthetically labeloligonucleotides with myriad different rhodamines, obviating the needfor laborious post-synthesis modifications. The use of an exemplarysynthesis reagents to synthesize an oligonucleotide labeled with an NHrhodamine dye is illustrated in FIG. 9 .

As will be appreciated by skilled artisans, owing to the availability ofphosphoramidite reagents that can act as donors, acceptors, or evenquenchers for NH-rhodamine dyes, the reagents described herein permitthe ability to synthesize oligonucleotides labeled with energy transferdyes and/or NH-rhodamine-quencher dye pairs, that are synthesized insitu. Exemplary syntheses of oligonucleotides labeled withNH-rhodamine-fluorescein energy transfer dye pairs that illustrate theversatility provided by the reagents described herein are illustrated inFIGS. 10 and 11 . Because the reagents described herein permit virtuallyany NH-rhodamine dye to be included in a solid support and/or synthesisreagent, oligonucleotides labeled with energy transfer dye pairs havingspectral properties that are adjusted for specified applications can beconveniently synthesized in situ, without the need for post synthesismodification. Moreover, oligonucleotides labeled with myriad differentenergy transfer dye pair combinations can be synthesized from individualmonomer reagents, obviating the need to make synthesis reagentscontaining specified dye pairs. Each member of the dye pair can beattached to the nascent oligonucleotide in a step-wise fashion, with orwithout the addition of intervening linking moieties.

Referring to FIG. 9 , support-bound synthetic oligoncleotide 200 istreated with acid to remove the DMT group protecting its 5′-hydroxyl,yielding 5′-deprotected support-bound oligonucleotide 202. Coupling ofN-protected NH-rhodamine phosphoramidite reagent 204 followed byoxidation yields support-bound NH-rhodamine-labeled oliognucleotide 206.Treatment with concentrated ammonium hydroxide to remove any protectinggroups and cleave the synthesized oligonucleotide from the solid support(resin) yields an oligonucleotide 208 that is labeled with anNH-rhodamine dye.

Referring to FIG. 10 , solid support reagent 210, which includes aprotected NH-rhodamine-fluorescein energy transfer dye pair as the labelmoiety, can undergo three cycles of synthesis to yield labeledsupport-bound oligonucleotide 212. Cleavage from the solid supportyields deprotected, labeled oligonucleotide 214.

Referring to FIG. 11A, nascent support-bound oligonucleotide 220 can belabeled with an NH-rhodamine-fluorescein dye pair synthesized in situ bycoupling N-protected NH-rhodamine phosphoramidite synthesis reagent 222to the 5′-hydroxyl of oligonucleotide 220, which, after oxidation,yields NH-rhodamine-labeled oligonucleotide 224. Removal of the DMTgroup followed by coupling with an O-protected phosphoramidite (which inthe specific example illustrated is FAM-phosphoramidite) yields labeled,support-bound oligonucleotide 226. Cleavage and deprotection yieldsoligonucleotide 228, which is labeled with an NH-rhodamine-FAM energytransfer dye pair.

The length and character of the linkage linking the donor and acceptordyes can also be manipulated through the use of phosphoramidite linkerreagents. This aspect is illustrated in FIG. 11B, where linkerphosphoramidite 230 is coupled to rhodamine-labeled oligonucleotide 225,yielding reagent 232. Coupling with FAM-phosphoramidite followed byoxidation, deprotection and cleavage yields oligonucleotide 234, whichis labeled with an NH-rhodamine-FAM energy transfer dye pair. In linkerphosphoramidite 230, “Sp” is a spacer, as previously defined. Forexample, “Sp” could represent (Sp.1), (Sp.2), (Sp.3), (Sp.4) or (Sp.5),as previously defined.

In the scheme illustrated in FIG. 11B, the length and properties of thelinker linking the NH-rhodamine and FAM dyes can be adjusted by couplingadditional linker phosphoramidites to reagent 232 prior to coupling withthe FAM-phosphoramidite. The linker phosphosphoramites could be thesame, or they could be different. In this way, oligonucleotides labeledwith energy transfer dye pairs in which the donor and acceptor dyes, aswell as the linker linking the donor and acceptors, are tailored forspecific purposes can be readily synthesized in situ.

While FIGS. 11A & B exemplify the use of a specific N-protectedNH-rhodamine reagent, skilled artisan will appreciate that anyN-protected NH-rhodamine reagent that acts as an acceptor for FAM couldbe used. Moreover, other O-protected fluoresceins could be used, ascould other types of phosphormidite dyes. Since the dyes are added asmonomers, the number of energy transfer dye labels available is greaterthan the number of phosphoramidite reagents necessary to synthesizethem. For example, oligonucleotides labeled with 9 differentenergy-transfer dye pairs can be synthesized from 3 differentN-protected NH-rhodamine phosphoramidite reagents (reagents A, B and C)and 3 different O-protected fluorescein phosphoramidite reagents(reagents 1, 2 and 3): oligo-A1, oligo-A2, oligo-A3, oligo-B1, oligo-B2,oligo-B3, oligo-C1, oligo-C2 and oligo-C3.

6. EXAMPLES Example 1 Synthesis of N-Protected NH-RhodaminePhosphoramidite Synthesis Reagents

Parent NH-rhodamine dyes including a carboxyl substitutent at the C5- orC6-position were synthesized as described in U.S. Pat. Nos. 4,622,400,5,750,409, 5,847,162, 6,017,712, 6,080,852, 6,184,379 or 6,248,884. Theparent NH-rhodamine dyes were then protected at the exocylcic amineswith either acetyl or trifluoroacetyl protecting groups, the resultantN-protected NH-rhodamine dyes were converted to hydroxyl-amidederivatives via the corresponding NHS ester derivative and the hydroxylfunctionality converted to a phosphoramidite using standard procedures.The overall scheme is llustrated below:

Protection with acetyl groups. NH-rhodamine dye acid 6 (mono TEA salt,1.676 mmol) was suspended in DCM (40 mL) and TEA (3.67 mL). Aceticanhydride (3.13 mL) was added, and the reaction mixture was stirred atroom temperature for 3 days. H₂O (10 mL) was added and stirring wascontinued for 30 min. The mixture was diluted with DCM (200 mL), washedwith NaHCO₃ solution (200 mL×2), dried (Na₂SO₄), filtered, andevaporated. The residue was purified by flush chromatography on silica(using a gradient of MeOH/EtOAc/DCM as eluent, from 5:20:75 to20:20:60). Evaporation of the appropriate fractions gave 881 mg (87%) ofcolorless lactonized bis acetyl N-protected NH-rhodamine dye 8.

Protection with trifluoroacetyl groups. NH-rhodamine dye acid 6 (monoTEA salt, 1.5 g, 2.402 mmol) was suspended in DCM (30 mL) and TEA (6.696mL). The suspension was cooled to 0° C. and trifluoroacetic anhydride(2.0 mL) added drop wise through a syringe. After the addition wascomplete, the reaction mixture was stirred at room temperature for 10min (and sonicated to break up dye particles). The resultant brownishsolution was evaporated, re-dissolved in DCM (50 mL), and stirred with5% HCl (40 mL) at room temperature for 1 h. The reaction mixture wastransferred to a separatory funnel and the two layers separated. The DCMlayer was washed with a Brine solution (40 mL), dried (Na₂SO₄),filtered, and evaporated. The residue was co-evaporated with MeCN (2×)to give 1.79 g of crude N-protected NH-rhodamine dye 7.

Synthesis of N-Protected NH-Rhodamine NHS Esters. To a solution ofbis-acetyl dye 8 (588 mg, 0.968 mmol) and NHS (334 mg, 2.904 mmol) inDCM (20 mL), was added DCC (599 mg, 2.904 mmol). The reaction mixturewas stirred at room temperature for 2 h, and then the reaction mixturewas filtered. The filtrate was diluted with DCM (80 mL), washed with H₂O(50 mL×2), dried (Na₂SO₄), filtered, and evaporated. The residue waspurified by flash chromatography on silica (using a gradient ofAcOH/MeOH/EtOAc/DCM as eluent, from 1:1:20:78 to 1:5:20:74). Evaporationof the appropriate fractions gave 680 mg (100%) of bis acetylN-protected NH-rhodamine NHS ester 10.

Following the procedure described above for bis acetyl N-protectedNH-rhodamine NHS ester 10, bis-TFA N-protected NH-rhodamine Dye 7 (0.322mmol), gave bis-TFA N-protected NH-rhodamine NHS ester 9 in 80-90%yield.

Synthesis of N-Protected NH-Rhodamine Phosphoramidites. NHS ester 9 (0.4mmol) was dissolved in DCM (8 mL). To this stirred solution, a mixtureof 6-amino-1-hexanol/DIPEA/DCM (56 mg/0.07 mL/2 mL) was added. Thereaction was stirred at room temperature for 30 min. The solid byproductwas removed by filtration and the filtrate was purified by flushchromatography on silica (using a gradient of EtOAc/DCM as eluent, from30% to 60%). Evaporation of the appropriate fractions gave 0.344 mmol(86%) of bis-TFA-hexanolamide rhodamine dye 11.

NHS ester 10 was converted to the bis-acetyl-hexanolamide NH-rhodaminedye 12 using a similar procedure.

To a solution of bis-TFA-hexanolamide rhodamine dye 11 (0.338 mmol) and2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.214 mL, 0.674mmol, 2 equiv.) in DCM (10 mL), was added tetrazole amine (4 mg) in oneportion. The reaction mixture was stirred at room temperature for 20 hVolatile materials were removed by evaporation and the residue waspurified by precipitation three times from DCM/hexane. The solid productwas dried in vacuo to give 0.256 mmol (76%) bis-TFA-N-protectedNH-rhodamine dye phosphoramidite 13.

Similarly, bis-acetyl dye 12 was converted to phospharmidite 14.

Example 2 Synthesis of Heterodimeric Dye Networks

A dye network comprising an O-protected fluorescein linked to anN-protected NH-rhodamine was synthesized as illustrated below:

Bis-acetyl N-protected NH-rhodamine NHS ester 16 (324 mg, 0.460 mmol)was dissolved in a solution of DMF (8 mL) and DIPEA (0.3 mL).Fluoresecin derivative 17 (239 mg, 0.32 mmol; synthesized as describedin U.S. Pat. No. 5,800,996) was added and the reaction mixture stirredat room temperature for 1 h. The mixture was evaporated and thenco-evaporated with MeOH (2 ×). The residue was dissolved in 10% MeOH/DCM(100 mL) and washed with Brine solution (100 mL). The aqueous layer wasextracted with 10% MeOH/DCM (50 mL×3), and the combined organic layerwas dried (Na₂SO₄), filtered, and evaporated. The residue was purifiedby flush chromatography on silica (using a gradient of Me0H/DCM aseluent, from 10% to 30%). Evaporation of the appropriate fractions gave270 mg (63%) of heterodimeric dye network 19 (as the DIPEA salt).

The corresponding bis-TFA protected dye network 18 was synthesized by asimilar procedure.

Example 3 Synthesis of Heterodimeric Dye Network Phosphoramidite

A phosphoramidite synthesis reagent comprising the heterodimeric dyenetwork as the label moiety was synthesized as illustrated below:

A solution of heterodimeric dye network 19 (0.173 mmol), DIPEA (1.028mL) and pivalic anhydride (0.702 mL) in DCM (10 mL) was stirred at roomtemperature for 1 day. H₂O (5 mL) was added and stirring was continuedfor 1 h. The reaction mixture was diluted with DCM (50 mL) and washedwith H₂O (40 mL). The aqueous layer was extracted with DCM (40 mL). Thecombined organic layer was dried (Na₂SO₄), filtered, and evaporated. Theresidue was purified by flush chromatography on silica (using a gradientof MeOH/DCM as eluent, from 3% to 15%). Evaporation of the appropriatefractions gave 0.145 mmol (84%) of bis-acetyl bis-pivaloyl heterodimericdye derivative 21 (as the DIPEA salt).

Dye derivative 21 (0.146 mmol) was suspended in a dissolved of DIPEA(0.2 mL) and DCM (8 mL). Solid N-hydroxysuccinimide tetramethyluroniumtetrafluoroborate (88 mg) was added and the reaction stirred at roomtemperature for 1 h. 6-Amino-1-hexanol (51 mg) was added and stirringwas continued for 1 h. The reaction mixture was filtered and thefiltrate was diluted with DCM (50 mL). The DCM solution was washed withbrine solution (40 mL), dried (Na₂SO₄), filtered, and evaporated. Theresidue was purified by flush chromatography on silica gel (using agradient of MeOH:EtOAc:DCM as eluent, from 5:20:75 to 15:20:65).Evaporation of the appropriate fractions gave 0.124 mmol (85%) of Dyehexanolamide derivative 23 (as the DIPEA salt).

Dye derivative 23 (91 mg, 0.057 mmol) and tetrazole amine (2 mg) weredissolved in DCM (5 mL).2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (CE diamidite)(0.04 mL), and was added and the reaction stirred at room temperaturefor 16 h. The reaction mixture was diluted with a solution ofTEA:EtOAc:DCM (3:20:77, 3 mL) and purified by silica gel chromatography(using a gradient of TEA:MeOH:EtOAc:DCM as eluent, from 3:0:20:77 to3:5:20:72). Evaporation of the appropriate fractions and precipitationfrom DCM/hexane gave 75 mg (74%) of heterodimeric dye phosphoramidite25.

Example 4 Synthesis of a Non-Nucleosidic Phosphoramidite SynthesisReagent Including an Optional Synthesis Handle

A non-nucleosidic phosphoramidite synthesis reagent that includes anoptional synthesis handle of the formula —OR^(e) was synthesized asillustrated below:

Bis-TFA derivative 9 (1.354 mmol) was dissolved in a solution of DIPEA(0.236 mL) in DCM (30 mL). To the stirred solution, linker synthon 29and DCM (0.609 g, 1.355 mmol; synthesized as described in Nelson et al.,1992, Nucl. Acids Res. 20:6253-6259) was added stirring continued atroom temperature for 2 h. EtOAc was added and the mixture was loaded ona silica column. Pure product was isolated by elution using a gradientof EtOAc:DCM from 5:95 to 1:5. Evaporation of the appropriate fractionsgave 1.25 g (80%) of bis-TFA-rhodamine-hydroxy amide 30.

A solution of bis-TFA-rhodamine-hydroxy amide 30 (1.081 mmol) andtetrazole amine (18.5 mg) were dissolved in DCM (35 mL). CE diamiditewas added (0.685 mL) and the reaction stirred at room temperature for 18h. The solvent was removed under reduced pressure and the residueprecipitated from DCM/hexane (3×) to give 1.081 mmol (100%) of purebis-TFA-rhodamine DMT phosphoramidite 31.

Example 5 Solid Phase Synthesis of a Labeled Oligonucleotide

Oligonucleotides labeled with the N-protected NH-rhodaminephosphoramidite synthesis reagents were synthesized on polystyrene solidsupports using the standard operating conditions on an AB 3900 automatedDNA synthesizer. The N-protected NH-rhodamine phosphoramidites weresoluble in the acetonitrile solvent for the coupling reactions, and theN-protected Nh-rhodamine dye adducts were stable to repeated synthesiscycles which employed removal of DMT with dichloroacetic acid, additionof nucleoside phosphoramidite monomers, capping with acetic anhydrideand oxidation with iodine to generate the internucleotide phosphatelinkages. This class of NH-rhodamine was also found to be stable to theconditions used to deprotect and cleave the synthesized labeledoligonucleotide from the solid support (treatment with ammoniumhydroxide at 60° C. for 1-2 h). The overall scheme used to synthesizethe labeled oligonucleotide is illustrated below:

By this process, TFA-rhodamine DMT phosphoramidite 31 was coupled to the5′-hydroxyl of a support-bound oligonucleotide to give the phosphiteintermediate 32. Fluorescein phosphoramidite (Glenn Research) wascoupled to the free hydroxyl of intermediate 32. The resultant labeledoligo was oxidized, cleaved from the support with concentrated ammoniahydroxide for 1 to 2 hours at 60° C., washed with aceonitrile/water anddried under reduced pressure to yield labeled oligonucleoide 33. Oligo33 was re-precipitated using a standard sodium acetate/EtOHprecipitation protocol. Labeled oligo 33 was produced in greater then90% purity and in greater than 85% yield (170,000 pM from 0.2 uMsupport) and used without further purification.

Example 6 Labeled Oligonucleotides Synthesized with the SynthesisReagents Exhibit Good Spectral Properties

Poly(dT)₁₀ oligonucleotides labeled with NH-rhodamines orNH-rhodamine-fluorescein dye pairs were synthesized as illustratedabove. Following cleavage, fluorescence spectra were recorded. Alllabeled oligos synthesized exhibited good fluorescence properties.

Although the foregoing inventions have been described in some detail tofacilitate understanding, it will be apparent thatcertain changes andmodifications may be practiced within the scope of the appended claims.Accordingly, the described embodiments are to be considered asillustrative and not restrictive, and the various inventions describedherein are not to be limited to the details provided herein.

All literature and patent references cited throughout the disclosure areincorporated into the application by reference for all purposes.

1.-13. (canceled)
 14. A reagent useful for labeling an oligonucleotide,comprising a label moiety that comprises an N-protected NH-rhodaminemoiety, a phosphate ester precursor group, and a suitably protectedsynthesis handle, which is a compound according to structural formula(IX.1):

wherein LM represents the label moiety, —Z— represents a portion of alinkage contributed by a functional group F^(z), Sp¹, Sp² and Sp³, whichcan be the same or different, each represent spacing moieties, Grepresents CH, N, or a group comprising an arylene, phenylene,heteroarylene, lower cycloalkylene, cyclohexylene, and/or a lowercycloheteroalkylene, R^(e) represents an acid-labile protecting group,and PEP represents the phosphate ester precursor group.
 15. The reagentof claim 14 in which Sp¹, Sp² and Sp³ are each, independently of oneanother, selected from an alkylene chain containing from 1 to 10 carbonatoms, —(CH₂)_(a)—[(Ar)_(b)—(CH₂)_(a)]_(c)—,—(CH₂)_(a)—[C≡C—(CH₂)_(a)]_(c)—,—(CH₂)_(a)—[C≡C—(Ar)_(b)]_(c)—(CH₂)_(a)—, —(CH₂)_(d)—NH—C(O)—[(CH₂)_(a)—(Ar)—(CH₂)_(a)—C(O)—NH]_(c)—(CH₂)_(d)— and—[CH₂(CH₂)_(e)O]_(f)—CH₂(CH₂)—, where a, b, c, d, e, f and Ar are asdefined in claim
 4. 16. The reagent of claim 14 which is a compoundaccording to structural formula (IX.2), (IX.3), (IX.4) or (IX.5):

wherein B represents a suitably protected nucleobase, L² represents alinker linking label moiety LM to nucleobase B and, in structure (IX.4),R¹¹ represents a protecting group.
 17. The reagent of claim 16 in whichthe nucleobase is selected from adenine, 7-deazaguanine, guanine,7-deazaguanine, cytosine, uracil, thymine, inosine, xanthene andhypoxanthene.
 18. The reagent of claim 17 in which B is selected fromA^(iBu), A^(Pac), C^(Ac), G^(iPr-Pac), T and U.
 19. The reagent of claim16 in which L² is selected from —C≡C—CH₂—NH—, —C≡C—C(O)—, —CH═CH—NH—,—CH═CH—C(O)—, —C≡C—CH₂—NH—C(O)—(CH₂)₁₋₆—NH—,—CH═CH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —C≡CH—CH₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH—,—C≡C—C≡C—CH₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH—, —C≡C—(Ar)₁₋₂—C≡C—CH₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH—, —C≡C—(Ar)₁₋₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH— and—C≡C—(Ar)₁₋₂—O—CH₂CH₂—[O—CH₂CH₂]₀₋₆—NH—, where Ar is as defined in claim4.
 20. The reagent of claim 16 in which -B-L²- is selected from


21. (canceled)
 22. (canceled)
 23. (canceled)
 24. A reagent useful forlabeling an oligonucleotide, comprising a label moiety that comprises anN-protected NH-rhodamine moiety, a solid support, and a suitablyprotected synthesis handle.
 25. The reagent of claim 24, which is acompound according to structural formula (X):

wherein LM represents the label moiety, L represents an optionalselectively cleavable linker and R^(e) represents an acid-labileprotecting group.
 26. The reagent of claim 25 which is a compoundaccording to structural formula (XI.1):

wherein Z— represents a portion of a linkage contributed by a functionalgroup F^(z), Sp¹, Sp² and Sp³, which can be the same or different, eachrepresent spacing moieties, G represents CH, N, or a group comprising anarylene, phenylene, heteroarylene, lower cycloalkylene, cyclohexylene,and/or a lower cycloheteroalkylene, R^(e) represents an acid-labileprotecting group, and PEP represents the phosphate ester precursorgroup, and Sp⁴ represents a selectively cleavable spacing moiety.27.-36. (canceled)
 37. A reagent useful for labeling an oligonucleotide,comprising a label moiety that comprises an N-protected NH-rhodaminemoiety, in which the N-protected NH-rhodamine moiety comprises astructure selected from structural formulae (IIIc), (IIIb) and (IIIc):

wherein: R′ is selected from R^(3′) and hydrogen; R″ is selected fromR^(6′) and hydrogen; R⁹ is an aryl group of the formula —C(O)R¹⁰, whereR¹⁰ is selected from hydrogen, lower alkyl, methyl, —CX₃, —CHX₂, —CH₂X,—CH₂—OR^(d) and phenyl optionally mono-substituted with a lower alkyl,methyl, —X, —OR^(d), cyano or nitro group, where R^(d) is selected fromlower alkyl, phenyl and pyridyl, and each X is a halo group, typicallyfluoro, or chloro; R^(1′), R^(2′), R^(2″), R^(4′), R^(4″), R^(5′),R^(5″), R^(7′), R^(7″), R^(8′), R⁴, R⁵, R⁶, and R⁷, when taken alone,are each, independently of one another, selected from hydrogen, loweralkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20membered heteroarylalkyl, —R^(b) and —(CH₂)_(x)—R^(b), where x is anintger ranging from 1 to 10 and R^(b) is selected from —X, —OH, —OR^(a),—SH, —SR^(a), —NH₂, —NHR¹, —NR^(c)R^(c), —N⁺R^(c)R^(c)R^(c), perhalolower alkyl, trihalomethyl, trifluoromethyl, —B(OH)₃, —B(OR^(a))₃,—B(OH)O⁻, —B(OR^(a))₂O⁻, —B(OH)(O⁻)₂, —B(OR^(a))(O⁻)₂, —P(OH)₂, —O(OH)O⁻, —P(OR^(a))₂, —P(OR^(a))O⁻, —P(O)(OH)₂, —P(O)(OH)O⁻, —P(O)(O⁻)₂,—P(O)(OR^(a))₂, —P(O)(OR^(a))O⁻, —P(O)(OH)(OR^(a)), —OP(OH)₂, —OP(OH)O⁻,—OP(OR^(a))₂, —OP(OR^(a))O⁻, —OP(O)(OH)₂, —OP(O)(OH)O⁻, —O P(O)(O⁻)₂,—OP(O)(OR^(a))₂, —OP(O)(OR^(a))O⁻, —OP(O)(OR^(a))(OH), —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R^(a), —C(O) H, —C(O)R^(a), —C(S)X, —C(O)O⁻, —C(O)OH,—C(O)NH₂, —C(O)NHR^(a), —C(O)NR^(c)R^(c), —C(S)NH₂, —C(O)N HR^(a),—C(O)NR^(c)R^(c), —C(NH)NH₂, —C(NH)NHR^(a), and —C(NH)NR^(c)R^(c), whereX is halo, each R^(a) is, independently of the others, selected fromlower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryland 6-20 membered heteroarylalkyl, and each R^(c) is, independently ofthe others, an R^(a), or, alternatively, two R^(c) bonded to the samenitrogen atom may be taken together with that nitrogen atom to form a 5-to 8-membered saturated or unsaturated ring that may optionally includeone or more of the same or different ring heteroatoms, which aretypically selected from O, N and S or, alternatively, R^(1′) and R^(2′)or R^(7′) and R^(8′) are taken together with the carbon atoms to whichthey are bonded to form an optionally substituted (C6-C14) aryl bridgeand/or R^(4′) and R^(4″) and/or R^(5′) and R^(5″) are taken togetherwith the carbon atoms to which they are bonded to form a benzo group;and R^(3′) and R^(6′), when taken alone, are each, independently of oneanother, selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl,5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, oralternatively, R^(3′) and R^(2′) or R^(4′) and/or R^(6′) and R^(5′) orR^(7′) in the compounds of structural formula (IIIa), R^(3′) and R^(2′)or R^(4′) and/or R^(6′) and R^(5′) or R^(7″) in the compounds ofstructural formula (III6), or R^(3′) and R^(2″) or R^(4′) and/or R^(6′)and R^(5′) or R^(7″) in the compounds of structural formula (IIIc) aretaken together with the atoms to which they are bonded to form a 5- or6-membered saturated or unsaturated ring which is optionally substitutedwith one or more of the same or different lower alkyl, benzo or pyridogroups, with the proviso that at least one of R^(2′), R^(4′), R^(5′),R^(7′), R⁵ or R⁶ in the compounds structural formula (IIIa), at leastone of R^(2′), R^(4′), R^(5′), R^(7″), R⁵ or R⁶ in the compounds ofstructural formula (IIIb) and at least one of R^(2″), R^(4′), R^(5′),R^(7″), R⁵ or R⁶ in the compounds of structural formula (IIIc) comprisesa group of the formula —Y—, where Y represents a portion of a linkagecontributed by a functional group F^(y). 38.-66. (canceled)